Methods of treatment using specific binding agents of human angiopoietin-2

ABSTRACT

Disclosed are peptides that bind to Ang-2. Also disclosed are peptibodies comprising the peptides, methods of making such peptides and peptibodies, and methods of treatment using such peptides and peptibodies.

This application claims benefit as a divisional to U.S. application Ser.No. 10/410,998 filed Apr. 9, 2003, which is now U.S. Pat. No. 7,205,275,which is continuation in part of U.S. application Ser. No. 10/269,695filed Oct. 10, 2002, which is now U.S. Pat. No. 7,138,370, which in turnclaims benefit of U.S. Provisional Application Ser. No. 60/414,155,filed Sep. 27, 2002, and U.S. Provisional Application Ser. No.60/328,624 filed Oct. 11, 2001, all of which are incorporated herein byreference in their entirety.

FIELD OF INVENTION

The present invention relates to specific binding agents that recognizeand bind to angiopoietin-2 (Ang-2). More specifically, the inventionrelates to the production, diagnostic use, and therapeutic use of thespecific binding agents and fragments thereof, which specifically bindAng-2.

BACKGROUND OF THE INVENTION

Angiogenesis, the formation of new blood vessels from existing ones, isessential to many physiological and pathological processes. Normally,angiogenesis is tightly regulated by pro- and anti-angiogenic factors,but in the case of diseases such as cancer, ocular neovascular diseases,arthritis, and psoriasis, the process can go awry. Folkman, J., Nat.Med., 1:27-31 (1995).

Angiogenesis is believed to play an important role in sustaininginflammatory tissue expansion (pannus) in rheumatoid arthritis (Walsh etal., Arihritis Res., 3:147-153 (2001). In fact, there are a number ofdiseases known to be associated with deregulated or undesiredangiogenesis. See Carmeliet et al., Nature 407:249-257 (2000).

Such diseases include, but are not limited to, ocularneovascularisation, such as retinopathies (including diabeticretinopathy), age-related macular degeneration, psoriasis,hemangioblastoma, hemangipma, arteriosclerosis, inflammatory disease,such as a rheumatoid or rheumatic inflammatory disease, especiallyarthritis (including rheumatoid arthritis), or other chronicinflammatory disorders, such as chronic asthma, arterial orpost-transplantational atherosclerosis, endometriosis, and neoplasticdiseases, for example so-called solid tumors and liquid (orhematopoietic) tumors (such as leukemias and lymphomas). Other diseasesassociated with undesired angiogenesis will be apparent to those skilledin the art.

Although many signal transduction systems have been implicated in theregulation of angiogenesis, one of the best-characterized and mostendothelial cell-selective systems involves the Tie-2 receptor tyrosinekinase (referred to as “Tie-2” or “Tie-2R” (also referred to as “ORK”);murine Tie-2 is also referred to as “tek”) and its ligands, theangiopoietins (Gale, N. W. and Yancopoulos, G. D., Genes Dev.13:1055-1066 [1999]). There are 4 known angiopoietins; angiopoietin-1(“Ang-1”) through angiopoietin-4 (“Ang-4”). These angiopoietins are alsoreferred to as “Tie-2 ligands”. (Davis, S., et al., Cell, 87:1161-1169[1996]; Grosios, K., et al., Cytogenet Cell Genet, 84:118-120 [1999];Holash, J., et al., Investigative Ophthalmology & Visual Science,42:1617-1625 [1999]; Koblizek, T. I., et al., Current Biology, 8:529-532[1998]; Lin, P., et al., Proc Natl Acad Sci USA, 95:8829-8834 [1998];Maisonpierre, P. C., et al., Science, 277:55-60 [1997]; Papapetropoulos,A., et al., Lab Invest, 79:213-223 [1999]; Sato, T. N., et al., Nature,375:70-74 [1998]; Shyu, K. G., et al., Circulation, 98:2081-2087 [1998];Suri, C., et al., Cell, 87:1171-1180-[1996]; Suri, C., et al., Science,282:468471 [1998]; Valenzuela, D. M., et al., Proceedings of theNational Academy of Sciences of the USA, 96:1904-1909 [1999];Witzenbichler, B., et al., J Biol Chem, 273:18514-18521 [1998]). WhereasAng-1 binding to Tie-2 stimulates receptor phosphorylation in culturedendothelial cells, Ang-2 has been observed to both agonize andantagonize Tie-2 receptor phosphorylation (Davis, S., et al., [1996],supra; Maisonpierre, P. C., et al., [1997], supra; Kim, I., J. H. Kim,et al., Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P.C. Maisonpierre, et al., Cardiovascular Research 49(3): 659-70 (2001)).

The phenotypes of mouse Tie-2 and Ang-1 knockouts are similar andsuggest that Ang-1-stimulated Tie-2 phosphorylation mediates remodelingand stabilization of developing vessels in utero through maintenance ofendothelial cell-support cell adhesion (Dumont, D. J., et al., Genes &Development, 8:1897-1909 [1994]; Sato, T. N., et al., Nature, 376:70-74[1995]; Suri, C., et al., [1996], supra). The role of Ang-1 in vesselstabilization is thought to be conserved in the adult, where it isexpressed widely and constitutively (Hanahan, D., Science, 277:48-50[1997]; Zagzag, D., et al., Experimental Neurology, 159:391-400 [1999]).In contrast, Ang-2 expression is primarily limited to sites of vascularremodeling, where it is thought to block Ang-1 function, therebyinducing a state of vascular plasticity conducive to angiogenesis(Hanahan, D., [1997], supra; Holash, J., et al., Science, 284:1994-1998[1999]; Maisonpierre, P. C., et al., [1997], supra).

Numerous published studies have purportedly demonstratedvessel-selective Ang-2 expression in disease states associated withangiogenesis. These pathological conditions include, for example,psoriasis, macular degeneration, and cancer (Bunone, G., et al.,American Journal of Pathology, 155:1967-1976 [1999]; Etoh, T., et al.,Cancer Research, 61:2145-2153 [2001]; Hangai, M., et al., InvestigativeOphthalmology & Visual Science, 42:1617-1625 [2001]; Holash, J., et al.,[1999] supra; Kuroda, K., et al., Journal of Investigative Dermatology,116:713-720 [2001]; Otani, A., et al., Investigative Ophthalmology &Visual Science, 40:1912-1920 [1999]; Stratmann, A., et al., AmericanJournal of Pathology, 153:1459-1466 [1998]; Tanaka, S., et al., J ClinInvest, 103:34-345 [1999]; Yoshida, Y., et al., International Journal ofOncology, 15:1221-1225 [1999]; Yuan, K., et al., Journal of PeriodontalResearch, 35:165-171 [2000]; Zagzag, D., et al., [1999] supra). Most ofthese studies have focused on cancer, in which many tumor types appearto display vascular Ang-2 expression. In contrast with its expression inpathological angiogenesis, Ang-2 expression in normal tissues isextremely limited (Maisonpierre, P. C., et al., [1997], supra; Mezquita,J., et al., Biochemical and Biophysical Research Communications,260:492-498 [1999]). In the normal adult, the three main sites ofangiogenesis are the ovary, placenta, and uterus; these are the primarytissues in normal (i.e., non-cancerous) tissues in which Ang-2 mRNA hasbeen detected.

Certain functional studies suggest that Ang-2 may be involved in tumorangiogenesis. Ahmad et al. (Cancer Res., 61:1255-1259 [2001]) describeAng-2 over-expression and show that it is purportedly associated with anincrease in tumor growth in a mouse xenograft model. See also Etoh etal., supra, and Tanaka et al., supra, wherein data is presentedpurportedly associating Ang-2 over expression with tumorhypervascularity. However, in contrast, Yu et al. (Am. J. Path.,158:563-570 [2001]) report data to show that overexpression of Ang-2 inLewis lung carcinoma and TA3 mammary carcinoma cells purportedlyprolonged the survival of mice injected with the correspondingtransfectants.

In the past few years, various publications have suggested Ang-1, Ang-2and/or Tie-2 as a possible target for anticancer therapy. For example,U.S. Pat. Nos. 6,166,185, 5,650,490, and 5,814,464 each disclose theconcept of anti-Tie-2 ligand antibodies and receptor bodies. Lin et al.(Proc. Natl. Acad Sci USA, 95:8829-8834 [1998]) injected an adenovirusexpressing soluble Tie-2 into mice; the soluble Tie-2 purportedlydecreased the number and size of the tumors developed by the mice. In arelated study, Lin et al (J. Clin. Invest., 100:2072-2078 [1997])injected a soluble form of Tie-2 into rats; this compound purportedlyreduced tumor size in the rats. Siemeister et al. (Cancer Res.,59:3185-3189 [1999]) generated human melanoma cell lines expressing theextracellular domain of Tie-2, injected these cell lines into nude mice,and concluded that soluble Tie-2 purportedly resulted in a “significantinhibition” of tumor growth and tumor angiogenesis. In view of thisinformation, and given that both Ang-1 and Ang-2 bind to Tie-2, it isnot clear from these studies whether Ang-1, Ang-2, or Tie-2 would be anattractive target for anti-cancer therapy.

The fusion of certain peptides to a stable plasma protein such as an Igconstant region to improve the half-life of these molecules has beendescribed in, for example, PCT publication WO 00/24782, published May 4,2000.

The fusion of a protein or fragment thereof to a stable plasma proteinsuch as an Ig constant region to improve the half-life of thesemolecules has been variously described (see, for example, U.S. Pat. No.5,480,981; Zheng et al., J. Immunol., 154:5590-5600, (1995); Fisher etal., N. Engl. J. Med., 334:1697-1702, (1996); Van Zee, K. et al., J.Immunol., 156:2221-2230, (1996); U.S. Pat. No. 5,808,029, issued Sep.15, 1998; Capon et al., Nature, 337:525-531, (1989); Harvill et al.,Immunotech., 1:95-105, (1995); WO 97/23614, published Jul. 3, 1997;PCT/US 97/23183, filed Dec. 11, 1997; Linsley, J. Exp. Med.,174:561-569, (1991); WO 95/21258, published Aug. 10, 1995).

An effective anti-Ang-2 therapy might benefit a vast population ofcancer patients because most solid tumors require neovascularization togrow beyond 1-2 millimeters in diameter. Such therapy might have widerapplication in other angiogenesis-associated diseases as well, such asretinopathies, arthritis, and psoriasis.

There is an undeveloped need to identify new agents that specificallyrecognize and bind Ang-2. Such agents would be useful for diagnosticscreening and therapeutic intervention in disease states that areassociated with Ang-2 activity.

Accordingly, it is an object of the present invention to providespecific binding agents of Ang-2 that modulate Ang-2 activity. Suchagents of the present invention take the form of peptibodies, i.e.,peptides fused to other molecules such as an Fc domain of an antibody,where the peptide moiety specifically binds to Ang-2.

The disclosure all patents, patent applications, and other documentscited herein are hereby expressly incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

The present invention is directed in one embodiment to peptides (alsoreferred to as polypeptides herein) that bind to Ang-2. Also embodied inthe present invention are variants and derivatives of such peptides.

In another embodiment, the peptides and variants and derivatives thereofof the present invention are attached to vehicles.

In another embodiment, the peptides may be fused to Fc domains, therebyproviding peptibodies. Optionally, the peptibodies comprise at least onepeptide of, for example, SEQ ID NO:3-SEQ ID NO:6, or SEQ ID NO:76-SEQ IDNO:157, as well as variants and derivatives thereof. Further, thepeptides may comprise at least one peptide according to the formulae setforth in SEQ ID NO:65-SEQ ID NO:75, and SEQ ID NO:158.

In yet another embodiment, the invention provides. nucleic acidmolecules encoding the specific binding agents, and variants andderivatives thereof.

In still another embodiment, the invention provides nucleic acidmolecules encoding the peptibodies, as well as variants and derivativesthereof. Optionally, such nucleic acid molecules include SEQ IDNO:33-SEQ ID NO:53.

In still another embodiment, the invention provides a method ofdecreasing a tumor by administering an effective amount of the specificbinding agents of the present invention to a subject in need thereof.The invention also provides a method of inhibiting angiogenesis in asubject, comprising administering an effective amount of the specificbinding agents of the present invention to a subject in need thereof.The invention further provides a method of treating cancer in a subject,comprising an effective amount of the specific binding agents of thepresent invention to a subject in need thereof.

The invention also relates to a polypeptide capable of binding Ang-2wherein the polypeptide comprises the amino acid sequence WDPWT (SEQ IDNO: 65), and wherein the polypeptide is from about 5 to about 50 aminoacids in length, as well as physiologically acceptable salts thereof.The polypeptide can also comprise the amino acid sequence:

WDPWTC (SEQ ID NO: 66)and physiologically acceptable salts thereof. Additionally, thepolypeptide can comprise the amino acid sequence:

Cz ² WDPWT (SEQ ID NO: 67)wherein z² is an acidic or neutral polar amino acid residue, andphysiologically acceptable salts thereof. The polypeptide can furthercomprise the amino acid sequence:

Cz ² WDPWTC (SEQ ID NO: 68)wherein z² is an acidic or neutral polar amino acid residue, andphysiologically acceptable salts thereof.

In another embodiment, the invention relates to a polypeptide capable ofbinding Ang-2 comprising an amino acid sequence of the formula:

a¹a²a³ Ca ⁵ WDPWTCa¹²a¹³a¹⁴ (SEQ ID NO: 69)wherein:

a¹, a², and a³ are each independently amino acid residues;

a⁵ is an amino acid residue;

a¹² is absent or an amino acid residue;

a¹³ is absent or a neutral hydrophobic, neutral polar, or a basic aminoacid residue;

a¹⁴ is a neutral hydrophobic or neutral polar amino acid residue;

and physiologically acceptable salts thereof. In a preferred embodiment:

a¹ is V, I, P, W, G, S, Q, N, E, K, R, or H;

a² is V, P, M, G, S, Q, D, E, K, R, or H;

a³ is A, V, P, M, F, T, G, D, E, K, or H;

a⁵ is A, V, G, Q, N, D, or E;

a¹² is S, Q, N, D, E, K, or R;

a¹³ is L, T, or H; and

a a¹⁴ is V, L, I, W, or M.

In a more preferred embodiment, a¹ is Q; a² is E; a³ is E; a⁵ is D or E;a¹² is D or E; a¹³ is H; and a¹⁴ is M.

It will be appreciated that the use of lower case letters withsuperscripted numbers herein (such as a¹ and b¹) are intended toidentify amino acid positions, and are not meant to indicate the singleletter abbreviations for a given amino acid. Single letter amino acidabbreviations are given in upper case letters herein.

The invention further relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:

(SEQ ID NO: 70) b¹b²b³b⁴b⁵b⁶ Cb ⁸ WDPWTCb¹⁵b¹⁶b¹⁷b¹⁸b¹⁹b²⁰wherein:

b¹ is absent or an amino acid residue;

b² is absent or a neutral hydrophobic, neutral polar, or a basic aminoacid residue;

b³, b⁴, b⁵, and b⁶ are each independently absent or amino acid residues;

b⁸ is an amino acid residue;

b¹⁵ is absent or an amino acid residue;

b¹⁶ is absent or a neutral hydrophobic, neutral polar, or a basic aminoacid residue;

b¹⁷ is absent or a neutral hydrophobic or neutral polar amino acidresidue;

b¹⁸, b¹⁹, and b²⁰ are each independently absent or amino acid residues;

and physiologically acceptable salts thereof. In a preferred embodiment:

-   -   b¹ is absent, or A, V, L, P, W, F, T, G, S, Q, N, K, R, or H;    -   b² is absent, or A, V, L, I, P, W, M, T, G, S, Y, N, K, R, or H;    -   b³ b is absent, or A, L, I, P, W, M, T, G, S, Q, N, E, R, or H;    -   b⁴ is V, I, P, W, G, S, Q, N, E, K, R, or H;    -   b⁵ is V, P, M, G, S, Q, D, E, K, R, or H;    -   b⁶ is A, V, P, M, F, T, G, D, E, K, or H;    -   b⁸ is A, V, G, Q, N, D, or E;    -   b¹⁵ is S, Q, N, D, E, K, or R;    -   b¹⁶ is L, T, or H;    -   b¹⁷ is V, L, I, W, or M;    -   b¹⁸ is absent, or A, V, L, P, W, F, T, G, Y, Q, D, E, or R;    -   b¹⁹ is absent, or V, L, I, P, T, G, S, Y, Q, N, D, E, or R; and    -   b²⁰ is absent, or V, L, P, W, M, T, G, S, Y, Q, N, D, K, or R.

In a more preferred embodiment, b¹ is absent, or P, or T; b² is absent,or I, or N; b³ is absent, or R, or I; b⁴ is Q; b⁵ is E; b⁶ is E; b⁸ is Dor E; b¹⁵ is D or E; b¹⁶ is H; b¹⁷ is M; b¹⁸ is absent, or W, or P; b¹⁹is absent, or G, or E; and b²⁰ is absent, or V, or K.

It will also be appreciated that the invention preferably relates to apolypeptide comprising at least one amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, and SEQ ID NO: 76 to SEQ ID NO:118, inclusive, wherein the polypeptide is capable of binding to Ang-2,as well as physiologically acceptable salts thereof. The peptidesequences are set forth below:

TABLE 1 PEPTIDE SEQ ID NO. PEPTIDE SEQUENCE Con4-44 76PIRQEECDWDPWTCEHMWEV Con4-40 77 TNIQEECEWDPWTCDHMPGK Con4-4 78WYEQDACEWDPWTCEHMAEV Con4-3 1 79 NRLQEVCEWDPWTCEHMENV Con4-C5 80AATQEECEWDPWTCEHMPRS Con4-42 81 LRHQEGCEWDPWTCEHMFDW Con4-35 82VPRQKDCEWDPWTCEHMYVG Con4-43 83 SISHEECEWDPWTCEHMQVG Con4-49 84WAAQEECEWDPWTCEHMGRM Con4-27 85 TWPQDKCEWDPWTCEHMGST Con4-48 86GHSQEECGWDPWTCEHMGTS Con4-46 87 QHWQEECEWDPWTCDHMPSK Con4-41 88NVRQEKCEWDPWTCEHMPVR Con4-36 89 KSGQVECNWDPWTCEHMPRN Con4-34 90VKTQEHCDWDPWTCEHMREW Con4-28 91 AWGQEGCDWDPWTCEHMLPM Con4-39 92PVNQEDCEWDPWTCEHMPPM Con4-25 93 RAPQEDCEWDPWTCAHMDIK Con4-50 94HGQNMECEWDPWTCEHMFRY Con4-38 95 PRLQEECVWDPWTCEHMPLR Con4-29 96RTTQEKCEWDPWTCEHMESQ Con4-47 97 QTSQEDCVWDPWTCDHMVSS Con4-20 98QVIGRPCEWDPWTCEHLEGL Con4-45 99 WAQQEECAWDPWTCDHMVGL Con4-37 100 LPGQEDCEWDPWTCEHMVRS Con4-33 101  PMNQVECDWDPWTCEHMPRS AC2-Con4 102 FGWSHGCEWDPWTCEHMGST Con4-32 103  KSTQDDCDWDPWTCEHMVGP Con4-17 104 GPRISTCQWDPWTCEHMDQL Con4-8 105  STIGDMCEWDPWTCAHMQVD AC4-Con4 106 VLGGQGCEWDPWTCRLLQGW Con4-1 107  VLGGQGCQWDPWTCSHLEDG Con4-C1 108 TTIGSMCEWDPWTCAHMQGG Con4-21 109  TKGKSVCQWDPWTCSHMQSG Con4-C2 110 TTIGSMCQWDPWTCAHMQGG Con4-18 111  WVNEVVCEWDPWTCNHWDTP Con4-19 112 VVQVGMCQWDPWTCKHMRLQ Con4-16 113  AVGSQTCEWDPWTCAHLVEV Con4-11 114 QGMKMFCEWDPWTCAHIVYR Con4-C4 115  TTIGSMCQWDPWTCEHMQGG Con4-23 116 TSQRVGCEWDPWTCQHLTYT Con4-15 117  QWSWPPCEWDPWTCQTVWPS Con4-9 118 GTSPSFCQWDPWTCSHMVQG TN8-Con4*  4 QEECEWDPWTCEHM

It will be appreciated that certain peptides and/or peptibodies maycontain the prefix “TN”, “TN8”, or “TN12”, and that this prefix may ormay not be present for a given peptibody. Thus, for example, the terms“TN8-Con4” and “Con4” are used interchangeably herein.

In another embodiment, the invention relates to a composition of matterhaving the formula:(X ¹)_(a)-F¹-(X ²)_(b)

and multimers thereof, wherein:

F¹ is a vehicle;

X¹ and X² are each independently selected from

-   -   -(L¹)_(c)-P¹;    -   -(L¹)_(c)-P¹-(L²)_(d)-P²;    -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and    -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;

wherein one or more of P¹, P², P³, and P⁴ each independently comprise apolypeptide as described herein. For example, in a preferred embodiment,P¹, P², P³, and P⁴ can each independently comprise a polypeptide of SEQID NO: 3 to SEQ ID NO: 6, and/or SEQ ID NO: 76 to SEQ ID NO: 157.

In another embodiment, the composition of matter is of the formulae:X¹-F¹orF¹-X²and physiologically acceptable salts thereof, where X¹, F¹, and X² areas defined herein. In another embodiment, the composition of matter isof the formula:F¹-(L¹)_(c)-P¹and physiologically acceptable salts thereof, where L¹, F¹, and P¹ areas defined herein. In yet another embodiment, the composition of matteris of the formula:F¹-(L¹)_(c)-P¹-(L²)_(d)-P²and physiologically acceptable salts thereof, where L¹, F¹, P¹, P², andc and d are as defined herein. In still another embodiment thecomposition of matter is of the formula:P¹-(L¹)_(c)-F¹-(L²)_(d)-P²and physiologically acceptable salts thereof. In a preferred embodiment,F¹ is an Fc domain or fragment thereof.

The invention further relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:

Pc² Dc⁴ Lc⁶c⁷c⁸ LY (SEQ ID NO: 71)wherein

c² is a neutral hydrophobic amino acid residue

c⁴ is a A, D, or E

c⁶ is an acidic amino acid residue

c⁷ is an amino acid residue; and

c⁸ is a neutral hydrophobic, neutral polar, or basic amino acid residue;

and physiologically acceptable salts thereof. In a preferred embodiment,c² is L or M. In another preferred embodiment, c⁶ is D or E.

The invention further relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:

(SEQ ID NO: 72) d¹d²d³d⁴ Pd⁶ Dd⁸ Ld¹⁰d¹¹d¹² LYd¹⁵d¹⁶d¹⁷d¹⁸d¹⁹d²⁰d²¹d²²wherein,

d¹ is absent, or an amino acid residue;

d² is absent, or a neutral polar, acidic, or a basic amino acid residue;

d³ is absent, or a neutral hydrophobic or neutral polar amino acidresidue;

d⁴ is absent, or an amino acid residue;

d⁶ is a neutral hydrophobic amino acid residue;

d⁸ is a A, D, or E;

d¹⁰ is an acidic amino acid residue;

d¹¹ is an amino acid residue;

d¹² is a neutral hydrophobic, neutral polar, or basic amino acidresidue;

d¹⁵ is absent, or a neutral polar, acidic, or a basic amino acidresidue;

d¹⁶ is absent, or a neutral polar, acidic, or a basic amino acidresidue;

d¹⁷ is absent, or a neutral hydrophobic, or neutral polar amino acidresidue;

d¹⁸ is absent, or a neutral hydrophobic, or neutral polar amino acidresidue;

d¹⁹ is absent, or a neutral hydrophobic, neutral polar, or basic aminoacid residue;

d²⁰ is absent, or an amino acid residue;

d²¹ is absent, or a neutral polar, acidic, or a basic amino acidresidue;

d²² is absent, or a neutral hydrophobic, neutral polar, or basic aminoacid residue;

and physiologically acceptable salts thereof. In a preferred embodiment:

d¹ is T, S, Q, R, or H;

d² is T, Q, N, or K;

d³ is F;

d⁴ is M, Q, E, or K;

d⁶ is L or M;

d⁸ is D or E;

d¹⁰ is E;

d¹¹ is Q or E;

d¹² is T or R;

d¹⁵ Y, D, E, or K;

d¹⁶ is Q;

d¹⁷ is W or F;

d¹⁸ is L, I, M, or T;

d¹⁹ is L, F, or Y;

d²⁰ is Q, D, or E;

d²¹ is absent, Q, or H;

d²² is absent, A, L, G, S, or R.

In a preferred embodiment, the polypeptide comprises at least one aminoacid sequence selected from the group consisting of SEQ ID NO: 6, andSEQ ID NO: 119 to SEQ ID NO: 142, inclusive, wherein the polypeptide iscapable of binding to Ang-2. SEQ ID NO: 6, and SEQ ID NOS: 119-142 areset forth below:

Peptide SEQ ID NO. Peptide Sequence L1-1 119 QNYKPLDELDATLYEHFIFHYT L1-2120 LNFTPLDELEQTLYEQWTLQQS L1-3 121 TKFNPLDELEQTLYEQWTLQHQ L1-4 122VKFKPLDALEQTLYEHWMFQQA L1-5 123 VKYKPLDELDEILYEQQTFQER L1-7 124TNFMPMDDLEQRLYEQFILQQG L1-9 125 SKFKPLDELEQTLYEQWTLQHA L1-10 126QKFQPLDELEQTLYEQFMLQQA L1-11 127 QNFKPMDELEDTLYKQFLFQHS L1-12 128YKFTPLDDLEQTLYEQWTLQHV L1-13 129 QEYEPLDELDETLYNQWMFHQR L1-14 130SNFMPLDELEQTLYEQFMLQHQ L1-15 131 QKYQPLDELDKTLYDQFMLQQG L1-16 132QKFQPLDELEETLYKQWTLQQR L1-17 133 VKYKPLDELDEWLYHQFTLHHQ L1-18 134QKFMPLDELDEILYEQFMFQQS L1-19 135 QTFQPLDDLEEYLYEQWIRRYH L1-20 136EDYMPLDALDAQLYEQFTLLHG L1-21 137 HTFQPLDELEETLYYQWLYDQL L1-22 138YKFNPMDELEQTLYEEFLFQHA AC6-L1 139 TNYKPLDELDATLYEHWILQHS L1-C1 140QKFKPLDELEQTLYEQWTLQQR L1-C2 141 TKFQPLDELDQThYEQWTLQQR L1-C3 142TNFQPLDELDQTLYEQWTLQQR L1   6 KFNPLDELEETLYEQFTFQQ

The invention also relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:)

RPe³e⁴e⁵e⁶e⁷ G (SEQ ID NO: 73)wherein

e³ is a neutral polar amino acid residue;

e⁴ is an acidic amino acid residue;

e⁵ is a neutral polar or an acidic amino acid residue;

e⁶ is a neutral hydrophobic amino acid residue;

e⁷ is a neutral hydrophobic amino acid residue;

and physiologically acceptable salts thereof. In a preferred embodiment,e³ is Y or C. In another preferred embodiment, e⁴ is D or E. In stillanother preferred embodiment, e⁶ is I or M.

The invention further relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:

(SEQ ID NO: 74) f¹f²f³f⁴ RPf⁷f⁸f⁹f¹⁰f¹¹ Gf¹³f¹⁴f¹⁵f¹⁶f¹⁷f¹⁸f¹⁹f²⁰wherein,

-   -   f¹ is a neutral hydrophobic or neutral polar amino acid residue;    -   f² is a neutral hydrophobic or neutral polar amino acid residue;    -   f³ is a neutral polar or acidic amino acid residue;    -   f⁴ is a neutral hydrophobic or neutral polar amino acid residue;    -   f⁷ is a neutral polar amino acid residue;    -   f⁸ is an acidic amino acid residue;    -   f⁹ is a neutral polar or acidic amino acid residue;    -   f¹⁰ is a neutral hydrophobic amino acid residue;    -   f¹¹ is a neutral hydrophobic amino acid residue;    -   f¹³ is a neutral hydrophobic or neutral polar amino acid        residue;    -   f¹⁴ is a neutral hydrophobic or neutral polar amino acid        residue;    -   f¹⁵ is a neutral polar amino acid residue;    -   f¹⁶ is a neutral polar amino acid residue;    -   f¹⁷ is a neutral polar or acidic amino acid residue;    -   f¹⁸ is a neutral hydrophobic or basic amino acid residue;    -   f¹⁹ is a neutral hydrophobic or neutral polar amino acid        residue; and    -   f²⁰ is a neutral hydrophobic or neutral polar amino acid        residue;        and physiologically acceptable salts thereof.

In a preferred embodiment:

-   -   f¹ is S, A, or G;    -   f² is G, Q, or P;    -   f³ is Q, G, or D;    -   f⁴ is L, M, or Q;    -   f⁷ is C or Y;    -   f⁸ is E or D;    -   f⁹ is E, G, or D;    -   f¹⁰ is I or M;    -   f¹¹ is F or L;    -   f¹³ is C or W;    -   f¹⁴ is G or P;    -   f¹⁵ T or N    -   f¹⁶ is Q, Y, or K;    -   f¹⁷ is N, D, or Q;    -   f¹⁸ is L, V, W, or R;    -   f¹⁹ is A, Q, Y, or I; and    -   f²⁰ is L, A, G, or V.

In a more preferred embodiment, the invention relates to a polypeptidecomprising at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 3, and SEQ ID NO: 143 to SEQ ID NO: 148,inclusive, wherein the polypeptide is capable of binding to Ang-2, andphysiologically acceptable salts thereof. SEQ ID NO: 3, and SEQ ID NO:143 to SEQ ID NO: 148 are as follows.

Peptide SEQ ID NO. Sequence Con 1-1 143 AGGMRPYDGMLGWPNYDVQA Con 1-2 144QTWDDPCMHILGPVTWRRCI Con 1-3 145 APGQRPYDGMLGWPTYQRIV Con 1-4 146SGQLRPCEEIFGCGTQNLAL Con 1-5 147 FGDKRPLECMFGGPIQLCPR Con 1-6 148GQDLRPCEDMFGCGTKDWYG Con 1   3 KRPCEEIFGGCTYQ

In still another aspect, the invention relates to a polypeptide capableof binding Ang-2 comprising an amino acid sequence of the formula:

Cg² Gg⁴g⁵ DPFTg¹⁰ GCg¹³ (SEQ ID NO: 75)wherein

-   -   g² is an acidic amino acid residue;

g⁴ is a neutral hydrophobic amino acid residue;

-   -   g⁵ is E, D, or Q;

g¹⁰ is a neutral hydrophobic or neutral polar amino acid residue;

-   -   g¹³ is an acidic residue;        and physiologically acceptable salts thereof. In a preferred        embodiment, g² is E or D. In another preferred embodiment, g⁴ is        V or M. In yet another embodiment, g¹⁰ is F or Q. In still        another embodiment, g¹³ is D or E.

The invention further relates to a polypeptide capable of binding Ang-2comprising an amino acid sequence of the formula:

(SEQ ID NO: 158) h¹h²h³h⁴ Ch⁶ Gh⁸h⁹ DPFTh¹⁴ GCh¹⁷h¹⁸h¹⁹h²⁰wherein,

-   -   h¹ is absent or a neutral hydrophobic, neutral polar, or a basic        amino acid residue;    -   h² is a neutral hydrophobic or neutral polar amino acid residue;    -   h³ is an acidic amino acid residue;    -   h⁴ is a neutral hydrophobic or neutral polar amino acid residue;    -   h⁶ is an acidic amino acid residue;    -   h⁸ is a neutral hydrophobic amino acid residue;    -   h⁹ is E, D, or Q;    -   h¹⁴ is a neutral hydrophobic or neutral polar amino acid        residue;

h¹⁷ is an acidic amino acid residue;

-   -   h¹⁸ is a neutral hydrophobic, neutral polar, or a basic amino        acid residue;    -   h¹⁹ is a neutral hydrophobic or neutral polar amino acid        residue; and    -   h²⁰ is absent or an amino acid residue;        and physiologically acceptable salts thereof.

In a preferred embodiment,

-   -   h¹ is absent, or A, L, M, G, K, or H;    -   h² is L, F, or Q;    -   h³ is D or E;    -   h⁴ is W or Y;    -   h⁶ is D or E;    -   h⁸ is V or M;

h¹⁴ is F or Q;

-   -   h¹⁷ is D or E;    -   h¹⁸ is M, Y, N, or K;

h¹⁹ is L or Q; and

-   -   h²⁰ is absent or M, T, G, S, D, K, or R.

In a more preferred embodiment, the invention relates to a polypeptidecomprising at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 5, or SEQ ID NO: 149 to SEQ ID NO: 157inclusive, wherein said polypeptide is capable of binding to Ang-2, andphysiologically acceptable salts thereof. SEQ ID NO: 5, and SEQ ID NO:149 to SEQ ID NO: 157 are set forth below.

Peptide SEQ ID NO: Sequence 12-9-1 149 GFEYCDGMEDPFTFGCDKQT 12-9-2 150KLEYCDGMEDPFTQGCDNQS 12-9-3 151 LQEWCEGVEDPFTFGCEKQR 12-9-4 152AQDYCEGMEDPFTFGCEMQK 12-9-5 153 LLDYCEGVQDPFTFGCENLD 12-9-6 154HQEYCEGMEDPFTFGCEYQG 12-9-7 155 MLDYCEGMDDPFTFGCDKQM 12-9-C2 156LQDYCEGVEDPFTFGCENQR 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR 12-9   5FDYCEGVEDPFTFGCDNH

In a highly preferred embodiment, the invention relates to a compositionof matter having the formula:(X¹)_(q)-F¹-(X²)_(r)

and multimers thereof, wherein:

F¹ is a vehicle;

X¹ and X² are each independently selected from

-   -   -(L¹)_(s)-P¹;    -   -(L¹)_(s)-P¹-(L²)_(t)-P²;    -   -(L¹)_(s)-P¹-(L²)_(t)-P²-(L³)_(u)-P³; and    -   -(L¹)_(s)-P¹-(L²)_(t)-P²-(L³)_(u)-P³-(L⁴)_(v)-P⁴;

wherein one or more of P¹, P², P³, and P⁴ each independently comprise apolypeptide selected from the group consisting of:

(a) the amino acid sequence WDPWT (SEQ ID NO: 65), wherein saidpolypeptide is from 5 to 50 amino acids in length;

(b) the amino acid sequence WDPWTC (SEQ ID NO: 66);

(c) the amino acid sequence Cz²WDPWT (SEQ ID NO: 67), wherein z² is anacidic or neutral polar amino acid residue;

(d) the amino acid sequence Cz²WDPWTC (SEQ ID NO: 68), wherein z² is anacidic or neutral polar amino acid residue;

(e) the amino acid sequence Pc²Dc⁴Lc⁶c⁷c⁸LY (SEQ ID NO: 71) wherein c²is a neutral hydrophobic amino acid residue; c⁴ is A, D, or E; c⁶ is anacidic amino acid residue; c⁷ is an amino acid residue; and c⁸ is aneutral hydrophobic, neutral polar, or basic amino acid residue;

(f) the amino acid sequence RPe³e⁴e⁵e⁶e⁷G (SEQ ID NO: 73) wherein e³ isa neutral polar amino acid residue; e⁴ is an acidic amino acid residue;e⁵ is a neutral polar or an acidic amino acid residue; e⁶ is a neutralhydrophobic amino acid residue; and e⁷ is a neutral hydrophobic aminoacid residue;

(g) the amino acid sequence Cg²Gg⁴g⁵DPFTg¹⁰GCg¹³ (SEQ ID NO: 75) whereing² is an acidic amino acid residue; g⁴ is a neutral hydrophobic aminoacid residue; g⁵ is a neutral polar or an acidic amino acid residue; g¹⁰is a neutral hydrophobic or neutral polar amino acid residue; and g¹³ isan acidic residue;

(h) A polypeptide of SEQ ID NO: 1;

(i) A polypeptide of SEQ ID NO: 2; and

(j) A polypeptide of SEQ ID NO: 7;

wherein L¹, L², L³, and L⁴ are each independently linkers; and q, r, s,t, u, and v are each independently 0 or 1, provided that at least one ofq and r is 1;

and physiologically acceptable salts thereof.

It will be appreciated that the invention further relates to a fusionpolypeptide comprising at least one peptide described as describedherein and a vehicle, wherein the fusion polypeptide is capable ofbinding to Ang-2, and physiologically acceptable salts thereof. In thefusion polypeptide, the vehicle is preferably at least one of an Fcdomain, polyethylene glycol, a lipid, a cholesterol group, acarbohydrate, and an oligosaccharide. Other suitable vehicles, such asalbumin and the like, will be appreciated by those skilled in the art,and are encompassed within the scope of the invention.

One skilled in the art will recognize that various molecules can beinserted into specific binding agent structure. Thus a given moleculecan be inserted, for example, between the peptide and vehicle portionsof the specific binding agents, or inserted within the peptide portionitself, while retaining the desired activity of specific binding agent.One can readily insert for example, molecules such as an Fc domain orfragment thereof, polyethylene glycol or other related molecules such asdextran, a fatty acid, a lipid, a cholesterol group, a smallcarbohydrate, a peptide, a cyotoxic agent, a chemotherapeutic agent, adetectable moiety as described herein (including fluorescent agents,radiolabels such as radioisotopes), an oligosaccharide, oligonucleotide,a polynucleotide, interference (or other) RNA, enzymes, hormones, or thelike. Other molecules suitable for insertion in this fashion will beappreciated by those skilled in the art, and are encompassed within thescope of the invention. This includes insertion of, for example, adesired molecule in between two consecutive amino acids, optionallyjoined by a suitable linker. By way of example, in the Con4(C) peptibodysequence:

M-Fc-GGGGGAQQEECEWDPWTCEHMLE (SEQ ID NO: 23)

one skilled in the art could readily insert a desired molecule between,for example, the two adjacent glutamine (“QQ”) residues to achieve adesired structure and/or function, while retaining the ability of thepeptide to bind Ang-2. Thus, this sequence could be modified as follows:

M-Fc-GGGGGAQ-[molecule]-QEECEWDPWTCEHMLE

Suitable linker molecules can be added if desired. It will further beappreciated that the molecule can be inserted in a number of locationson the molecule, including on suitable side chains, between the vehicleand peptide sequence as follows:

M-Fc-[molecule]-GGGGGAQQEECEWDPWTCEHMLE

or in any other location desired by one skilled in the art. Othersuitable embodiments will be apparent to those skilled in the art.

In still another embodiment, the invention relates to a polynucleotideencoding the specific binding agents (including, but not limited topeptides and/or peptibodies) of the invention, as described herein. Oneskilled in the art will appreciate that where the amino acid sequence isknown, the corresponding nucleotide sequence(s) can be readilydetermined using known techniques. See for example Suzuki, D., AnIntroduction to Genetic Analysis, W.H. Freeman Pub. Co. (1986).Exemplary nucleotide sequences encoding peptides of the invention areset forth below. One skilled in the art will recognize that more thanone codon can encode for a given amino acid, and therefore the inventionrelates to any nucleotide sequence which encodes the peptides and/orpeptibodies of the invention.

Seq. Peptide Id No. Peptide Sequence Exemplary DNA Sequence Con4-44  76PIRQEECDWDPWTCEHMWEV ccgatccgtcaggaagaatgcga ctgggacccgtggacctgcgaacacatgtgggaagtt (SEQ ID NO: 159) Con4-40  77 TNIQEECEWDPWTCDHMPGKaccaacatccaggaagaatgcga atgggacccgtggacctgcgacc acatgccgggtaaa (SEQ IDNO: 160) Con4-4  78 WYEQDACEWDPWTCEHMAEV tggtacgaacaggacgcttgcgaatgggacccgtggacctgcgaac acatggctgaagtt (SEQ ID NO: 161) Con4-31  79NRLQEVCEWDPWTCEHMIENV aaccgtctgcaggaagtttgcgaa tgggacccgtggacctgcgaacacatggaaaacgtt (SEQ ID NO: 162) Con4-C5  80 AATQEECEWDPWTCEHMPRSgctgctacccaggaagaatgcga atgggacccgtggacctgcgaac acatgccgcgttcc (SEQ IDNO: 163) Con4-42  81 LRHQEGCEWDPWTCEHMFDW ctgcgtcaccaggaaggttgcgaatgggacccgtggacctgcgaac acatgttcgactgg (SEQ ID NO: 164) Con4-35  82VPRQKDCEWDPWTCEHMYVG gttccgcgtcagaaagactgcga atgggacccgtggacctgcgaacacatgtacgttggt (SEQ ID NO: 165) Con4-43  83 SISHEECEWDPWTCEHMQVGtccatctcccacgaagaatgcgaa tgggacccgtggacctgcgaaca catgcaggttggt (SEQ IDNO: 360) Con4-49  84 WAAQEECEWDPWTCEHMGRM tgggctgctcaggaagaatgcgaatgggatccgtggacttgcgaaca catgggtcgtatg (SEQ ID NO: 166) Con4-27  85TWPQDKCEWDPWTCEHMGST acttggccgcaggacaaatgcga atgggatccgtggacttgcgaacacatgggttctact (SEQ ID NO: 167) Con4-48  86 GHSQEECGWDPWTCEHMGTSggtcactcccaggaagaatgcgg ttgggacccgtggacctgcgaac acatgggtacgtcc (SEQ IDNO: 168) Con4-46  87 QHWQEECEWDPWTCDHMPSK cagcactggcaggaagaatgcgaatgggacccgtggacctgcgacc acatgccgtccaaa (SEQ ID NO: 169) Con4-41  88NVRQEKCEWDPWTCEHMPVR aacgttcgtcaggaaaaatgcgaa tgggacccgtggacctgcgaacacatgccggttcgt (SEQ ID NO: 170) Con4-36  89 KSGQVECNWDPWTCEHMPRNaaatccggtcaggttgaatgcaac tgggacccgtggacctgcgaaca catgccgcgtaac (SEQ IDNO: 171) Con4-34  90 VKTQEHCDWDPWTCEHMREW gttaaaacccaggaacactgcgactgggacccgtggacctgcgaac acatgcgtgaatgg (SEQ ID NO: 172) Con4-28  91AWGQEGCDWDPWTCEHMLPM gcttggggtcaggaaggttgcga ctgggacccgtggacctgcgaacacatgctgccgatg (SEQ ID NO: 173) Con4-39  92 PVNQEDCEWDPWTCEHMPPMccggttaaccaggaagactgcga atgggacccgtggacctgcgaac acatgccgccgatg (SEQ IDNO: 174) Con4-25  93 RAPQEDCEWDPWTCAHMDIK cgtgctccgcaggaagactgcgaatgggacccgtggacctgcgctc acatggacatcaaa (SEQ ID NO: 175) Con4-50  94HGQNMECEWDPWTCEHMFRY cacggtcagaacatggaatgcga atgggacccgtggacctgcgaacacatgttccgttac (SEQ ID NO: 176) Con4-38  95 PRLQEECVWDPWTCEHMPLRccgcgtctgcaggaagaatgcgtt tgggacccgtggacctgcgaaca catgccgctgcgt (SEQ IDNO: 177) Con4-29  96 RTTQEKCEWDPWTCEHMESQ cgtaccacccaggaaaaatgcgaatgggacccgtggacctgcgaac acatggaatcccag (SEQ ID NO: 178) Con4-47  97QTSQEDCVWDPWTCDHMVSS cagacctcccaggaagactgcgtt tgggacccgtggacctgcgaccacatggtttcctcc (SEQ ID NO: 179) Con4-20  98 QVIGRPCEWDPWTCEHLEGLcaggttatcggtcgtccgtgcgaa tgggacccgtggacctgcgaaca cctggaaggtctg (SEQ IDNO: 180) Con4-45  99 WAQQEECAWDPWTCDHMVGL tgggctcagcaggaagaatgcgcttgggacccgtggacctgcgacc acatggttggtctg (SEQ ID NO: 181) Con4-37 100LPGQEDCEWDPWTCEHMVRS ctgccgggtcaggaagactgcga atgggacccgtggacctgcgaacacatggttcgttcc (SEQ ID NO: 182) Con4-33 101 PMNQVECDWDPWTCEHMPRSccgatgaaccaggttgaatgcga ctgggacccgtggacctgcgaac acatgccgcgttcc (SEQ IDNO: 183) AC2-Con4 102 FGWSHGCEWDPWTCEHMGST ttcggttggtctcacggttgcgaatgggatccgtggacttgcgaacac atgggttctacc (SEQ ID NO: 184) Con4-32 103KSTQDDCDWDPWTCEHMVGP aaatccacccaggacgactgcga ctgggacccgtggacctgcgaacacatggttggtccg (SEQ ID NO: 185) Con4-17 104 GPRISTCQWDPWTCEHMDQLggtccgcgtatctccacctgccag tgggacccgtggacctgcgaaca catggaccagctg (SEQ IDNO: 186) Con4-8 105 STIGDMCEWDPWTCAHMQVD tccaccatcggtgacatgtgcgaatgggacccgtggacctgcgctca catgcaggttgac (SEQ ID NO: 187) AC4-Con4 106VLGGQGCEWDPWTCRLLQGW gttctgggtggtcagggttgcgaa tgggacccgtggacctgccgtctgctgcagggttgg (SEQ ID NO: 188) Con4-1 107 VLGGQGCQWDPWTCSHLEDGgttctgggtggtcagggttgccag tgggacccgtggacctgctccca cctggaagacggt (SEQ IDNO: 189) Con4-C1 108 TTIGSMCEWDPWTCAHMQGG accaccatcggttccatgtgcgaatgggacccgtggacctgcgctca catgcagggtggt (SEQ ID NO: 190) Con4-21 109TKGKSVCQWDPWTCSHMQSG accaaaggtaaatccgtttgccag tgggacccgtggacctgctcccacatgcagtccggt (SEQ ID NO: 191) Con4-C2 110 TTIGSMCQWDPWTCAHMQGGaccaccatcggttccatgtgccag tgggacccgtggacctgcgctca catgcagggtggt (SEQ IDNO: 192) Con4-18 111 WVNEVVCEWDPWTCNHWDTP tgggttaacgaagttgtttgcgaatgggacccgtggacctgcaaccac tgggacaccccg (SEQ ID NO: 193) Con4-19 112VVQVGMCQWDPWTCKHMRLQ gttgttcaggttggtatgtgccagt gggacccgtggacctgcaaacacatgcgtctgcag (SEQ ID NO: 194) Con4-16 113 AVGSQTCEWDPWTCAHLVEVgctgttggttcccagacctgcgaat gggacccgtggacctgcgctcac ctggttgaagtt (SEQ IDNO: 195) Con4-11 114 QGMLKMFCEWDPWTCAHIVYR cagggtatgaaaatgttctgcgaatgggacccgtggacctgcgctcac atcgtttaccgt (SEQ ID NO: 196) Con4-C4 115TTIGSMCQWDPWTCEHMQGG accaccatcggttccatgtgccag tgggacccgtggacctgcgaacacatgcagggtggt (SEQ ID NO: 197) Con4-23 116 TSQRVGCEWDPWTCQHLTYTacctcccagcgtgttggttgcgaat gggacccgtggacctgccagcac ctgacctacacc (SEQ IDNO: 198) Con4-15 117 QWSWPPCEWDPWTCQTVWPS cagtggtcctggccgccgtgcgaatgggacccgtggacctgccaga ccgtttggccgtcc (SEQ ID NO: 199) Con4-9 118GTSPSFCQWDPWTCSHMVQG ggtacctccccgtccttctgccagt gggacccgtggacctgctcccacatggttcagggt (SEQ ID NO: 200) TN8-Con4 4 QEECEWDPWTCEHMcaggaagaatgcgaatgggaccc atggacttgcgaacacatg (SEQ ID NO: 201) L1-1 119QNYKPLDELDATLYEHFIFHYT cagaactacaaaccgctggacga actggacgctaccctgtacgaacacttcatcttccactacacc (SEQ ID NO: 202) L1-2 120 LNFTPLDELEQTLYEQWTLQQSctgaacttcaccccgctggacgaa ctggaacagaccctgtacgaaca gtggaccctgcagcagtcc(SEQ ID NO: 203) L1-3 121 TKFNPLDELEQTLYEQWTLQHQ accaaattcaacccgctggacgaactggaacagaccctgtacgaac agtggaccctgcagcaccag (SEQ ID NO: 204) L1-4 122VKFKPLDALEQTLYEHWMFQQA gttaaattcaaaccgctggacgct ctggaacagaccctgtacgaacactggatgttccagcaggct (SEQ ID NO: 205) L1-5 123 VKYKPLDELDEILYEQQTFQERgttaaatacaaaccgctggacgaa ctggacgaaatcctgtacgaacag cagaccttccaggaacgt(SEQ ID NO: 206) L1-7 124 TNFMPMDDLEQRLYEQFILQQGaccaacttcatgccgatggacgac ctggaacagcgtctgtacgaaca gttcatcctgcagcagggt(SEQ ID NO: 207) L1-9 125 SKFKPLDELEQTLYEQWTLQHAtccaaattcaaaccgctggacgaa ctggaacagaccctgtacgaaca gtggaccctgcagcacgct(SEQ ID NO: 208) L1-10 126 QKFQPLDELEQTLYEQFMLQQAcagaaattccagccgctggacga actggaacagaccctgtacgaac agttcatgctgcagcaggct(SEQ ID NO: 209) L1-11 127 QNFKPMDELEDTLYKQFLFQHScagaacttcaaaccgatggacga attggaagacaccctgtacaaaca gttcctgttccagcactcc(SEQ ID NO: 210) L1-12 128 YKFTPLDDLEQTLYEQWTLQHVtacaaattcaccccgctggacgac ctggaacagaccctgtacgaaca gtggaccctgcagcacgtt(SEQ ID NO: 211) L1-13 129 QEYEPLDELDETLYNQWMFHQRcaggaatacgaaccgctggacga actggacgaaaccctgtacaacc agtggatgttccaccagcgt(SEQ ID NO: 212) L1-14 130 SNFMPLDELEQTLYEQFMLQHQtccaacttcatgccgctggacgaa ctggaacagaccctgtacgaaca gttcatgctgcagcaccag(SEQ ID NO: 213) L1-15 131 QKYQPLDELDKTLYDQFMLQQGcagaaataccagccgctggacga actggacaaaaccctgtacgatca gttcatgctgcagcagggt(SEQ ID NO: 214) L1-16 132 QKFQPLDELEETLYKQWTLQQRcagaaattccagccgctggacga actggaagaaaccctgtacaaac agtggaccctgcagcagcgt(SEQ ID NO: 215) L1-17 133 VKYKPLDELDEWLYHQFTLHHQgttaaatacaaaccgctggacgaa ctggacgaatggctgtaccacca gttcaccctgcaccaccag(SEQ ID NO: 216) L1-18 134 QKFMPLDELDEILYEQFMFQQScagaaattcatgccgctggacgaa ctggacgaaatcctgtacgaacag ttcatgttccagcagtccc(SEQ ID NO: 217) L1-19 135 QTFQPLDDLEEYLYEQWIRRYHcagaccttccagccgctggacga cctggaagaatacttgtacgaaca gtggatccgtcgttaccac(SEQ ID NO: 218) L1-20 136 EDYMPLDALDAQLYEQFILLHGgaagactacatgccgctggacgc tctggacgctcagctgtacgaaca gttcatcctgctgcacggt(SEQ ID NO: 219) L1-21 137 HTFQPLDELEETLYYQWLYDQLcacaccttccagccgctggacga actggaagaaaccctgtactacca gtggctgtacgaccagctg(SEQ ID NO: 220) L1-22 138 YKFNPMDELEQTLYEEFLFQHAtacaaattcaacccgatggacgaa ctggaacagaccctgtacgaaga attcctgttccagcacgct(SEQ ID NO: 221) AC6-L1 139 TNYKPLDELDATLYEHWILQHSaccaactacaaaccgctggacga actggacgctaccctgtacgaaca ctggatcctgcagcactcc(SEQ ID NO: 222) L1-C1 140 QKFKPLDELEQTLYEQWTLQQRcagaaattcaaaccgctggacga actggaacagaccctgtacgaac agtggaccctgcagcagcgt(SEQ ID NO: 223) L1-C2 141 TKFQPLDELDQTLYEQWTLQQRaccaaattccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt(SEQ ID NO: 224) L1-C3 142 TNFQPLDELDQTLYEQWTLQQRaccaacttccagccgctggacga actggaccagaccctgtacgaac agtggaccctgcagcagcgt(SEQ ID NO: 225) L1 6 KFNPLDELEETLYEQFTFQQ aaattcaacccgctggacgagctggaagagactctgtacgaacagttt acttttcaacag (SEQ ID NO: 226) Con1-1 143AGGMRPYDGMLGWPNYDVQA gctggtggtatgcgtccgtacgac ggtatgctgggttggccgaactacgacgttcaggct (SEQ ID NO: 227) Con1-2 144 QTWDDPCMHILGPVTWRRCIcagacttgggacgatccgtgcatg cacattctgggtccggttacttggc gtcgttgcatc (SEQ IDNO: 228) Con1-3 145 APGQRPYDGMLGWPTYQRIV gctccgggtcagcgtccgtacgacggtatgctgggttggccgaccta ccagcgtatcgtt (SEQ ID NO: 229) Con1-4 146SGQLRPCEEIFGCGTQNLAL tccggtcagctgcgtccgtgcgaa gaaatcttcggttgcggtacccagaacctggctctg (SEQ ID NO: 230) Con1-5 147 FGDKRPLECMFGGPIQLCPRttcggtgacaaacgtccgctggaa tgcatgttcggtggtccgatccag ctgtgcccgcgt (SEQ IDNO: 231) Con1-6 148 GQDLRPCEDMFGCGTKDWYG ggtcaggacctgcgtccgtgcgaagacatgttcggttgcggtaccaa agactggtacggt (SEQ ID NO: 232) 12-9-1 149GFEYCDGMEDPFTFGCDKQT ggtttcgaatactgcgacggtatg gaagacccgttcaccttcggttgcgacaaacagacc (SEQ ID NO: 233) 12-9-2 150 KLEYCDGMEDPFTQGCDNQSaaactggaatactgcgacggtatg gaagacccgttcacccagggttg cgacaaccagtcc (SEQ IDNO: 234) 12-9-3 151 LQEWCEGVEDPFTFGCEKQR ctgcaggaatggtgcgaaggtgttgaagacccgttcaccttcggttgc gaaaaacagcgt (SEQ ID NO: 235) 12-9-4 152AQDYCEGMEDPFTFGCEMQK gctcaggactactgcgaaggtatg gaagacccgttcaccttcggttgcgaaatgcagaaa (SEQ ID NO: 236) 12-9-5 153 LLDYCEGVQDPFTFGCENLDctgctggactactgcgaaggtgtt caggacccgttcaccttcggttgc gaaaacctggac (SEQ IDNO: 237) 12-9-6 154 HQEYCEGMEDPFTFGCEYQG caccaggaatactgcgaaggtatggaagacccgttcaccttcggttg cgaataccagggt (SEQ ID NO: 238) 12-9-7 155MLDYCEGMDDPFTFGCDKQM atgctggactactgcgaaggtatg gacgacccgttcaccttcggttgcgacaaacagatg (SEQ ID NO: 239) 12-9-C2 156 LQDYCEGVEDPFTFGCENQRctgcaggactactgcgaaggtgtt gaagacccgttcaccttcggttgc gaaaaccagcgt (SEQ IDNO: 240) 12-9-C1 157 LQDYCEGVEDPFTFGCEKQR ctgcaggactactgcgaaggtgttgaagacccgttcaccttcggttgc gaaaaacagcgt (SEQ ID NO: 241) 12-9   5FDYCEGVEDPFTFGCDNH ttcgactactgcgaaggtgttgaa gacccgttcactttcggctgtgata(SEQ ID NO: 242)

In still another embodiment, the invention relates to expression vectorscomprising at least one polynucleotide of the invention. In anotherembodiment, the invention relates to host cells comprising theexpression vector. It will be appreciated that the host cells arepreferably prokaryotic cells (such as E. coli cells) or eukaryoticcells.

The invention also relates to a pharmaceutical composition comprising aneffective amount of a composition as described herein, in admixture witha pharmaceutically acceptable carrier.

The invention also relates to a method of inhibiting undesiredangiogenesis in a mammal comprising administering a therapeuticallyeffective amount of a polypeptide or composition as described herein.The invention also relates to a method of modulating angiogenesis in amammal comprising administering a therapeutically effective amount of apolypeptide or composition as described herein. The invention furtherrelates to a method of inhibiting tumor growth characterized byundesired angiogenesis in a mammal comprising administering atherapeutically effective amount of a polypeptide or composition asdescribed herein. Additionally, the invention relates to a method oftreating cancer in a mammal comprising administering a therapeuticallyeffective amount of a polypeptide or composition as described herein,and a chemotherapeutic agent. In a preferred embodiment, thechemotherapeutic agent is at least one of 5-FU, CPT-11, and Taxotere. Itwill be appreciated, however, that other suitable chemotherapeuticagents and other cancer therapies can be used.

The invention also relates to a method of modulating at least one ofvascular permeability or plasma leakage in a mammal comprisingadministering a therapeutically effective amount of a polypeptide orcomposition as described herein. The invention further relates to amethod of treating at least one of ocular neovascular disease, obesity,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,inflammatory disorders, atherosclerosis, endometriosis, neoplasticdisease, bone-related disease, or psoriasis in a mammal comprisingadministering a therapeutically effective amount of a polypeptide orcomposition as described herein.

It will be appreciated that the specific binding agents of the inventioncan be used to treat a number of diseases associated with deregulated orundesired angiogenesis. Such diseases include, but are not limited to,ocular neovascularisation, such as retinopathies (including diabeticretinopathy and age-related macular degeneration) psoriasis,hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease,such as a rheumatoid or rheumatic inflammatory disease, especiallyarthritis (including rheumatoid arthritis), or other chronicinflammatory disorders, such as chronic asthma, arterial orpost-transplantational atherosclerosis, endometriosis, and neoplasticdiseases, for example so-called solid tumors and liquid tumors (such asleukemias). Additional diseases which can be treated by administrationof the specific binding agents will be apparent to those skilled in theart. Such additional diseases include, but are not limited to, obesity,vascular permeability, plasma leakage, and bone-related diseases,including osteoporosis. Thus, the invention further relates to methodsof treating these diseases associated with deregulated or undesiredangiogenesis.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein said peptide or peptibody comprises the aminoacid sequence:

WDPWT (SEQ ID NO: 65)

wherein said peptide is from about 5 to about 50 amino acids in length,and physiologically acceptable salts thereof.

In another embodiment, the peptide or peptibody may comprise the aminoacid sequence:

WDPWTC (SEQ ID NO: 66)and physiologically acceptable salts thereof.

In another embodiment, the peptide or peptibody comprises the amino acidsequence:

Cz ² WDPWT (SEQ ID NO: 67)

wherein z² is an acidic or neutral polar amino acid residue, andphysiologically acceptable salts thereof.

In still another embodiment, the peptide or peptibody comprises theamino acid sequence:

Cz ² WDPWTC (SEQ ID NO: 68)

wherein z² is an acidic or neutral polar amino acid residue, andphysiologically acceptable salts thereof.

In yet another embodiment, the peptide or peptibody comprises the aminoacid sequence:

a¹a²a³ Ca ⁵ WDPWTCa¹²a¹³a¹⁴ (SEQ ID NO: 69)

wherein:

-   -   a¹, a², and a³ are each independently amino acid residues;    -   a⁵ is an amino acid residue;    -   a¹² is absent or an amino acid residue;    -   a¹³ is absent or a neutral hydrophobic, neutral polar, or a        basic amino acid residue;    -   a¹⁴ is a neutral hydrophobic or neutral polar amino acid        residue;

and physiologically acceptable salts thereof. In a preferred embodiment,a¹ is V, I, P, W, G, S, Q, N, E, K, R, or H; a² is V, P, M, G, S, Q, D,E, K, R, or H; a³ is A, V, P, M, F, T, G, D, E, K, or H; a⁵ is A, V, G,Q, N, D, or E; a¹² is S, Q, N, D, E, K, or R; a¹³ is L, T, or H; a¹⁴ isV, L, I, W, or M. In a more preferred embodiment, a¹ is Q; a² is E; a³is E; a⁵ is D or E; a¹² is D or E; a¹³ is H; and a¹⁴ is M.Physiologically acceptable salts thereof are also suitable.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein the peptide or peptibody comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO: 4,and SEQ ID NO: 76 to SEQ ID NO: 118, inclusive, and physiologicallyacceptable salts thereof.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein said peptide or peptibody comprises the aminoacid sequence of the formula:

Pc² Dc⁴ Lc⁶c⁷c⁸ LY (SEQ ID NO: 71)

wherein: c² is a neutral hydrophobic amino acid residue; c⁴ is a A, D,or E; c⁶ is an acidic amino acid residue; c⁷ is an amino acid residue;and c⁸ is a neutral hydrophobic, neutral polar, or basic amino acidresidue; and physiologically acceptable salts thereof. In a preferredembodiment, c² is L or M. In another preferred embodiment, c⁶ is D or E.Physiologically acceptable salts thereof are also suitable.

In another embodiment, the peptide or peptibody comprises an amino acidsequence of the formula:

(SEQ ID NO: 72) d¹d²d³d⁴ Pd⁶ Dd⁸ Ld¹⁰d¹¹d¹² LYd¹⁵d¹⁶d¹⁷d¹⁸d¹⁹d²⁰d²¹d²²

wherein,

d¹ is absent, or an amino acid residue; d² is absent, or a neutralpolar, acidic, or a basic amino acid residue; d³ is absent, or a neutralhydrophobic or neutral polar amino acid residue; d⁴ is absent, or anamino acid residue; d⁶ is a neutral hydrophobic amino acid residue; d⁸is a A, D, or E; d¹⁰ is an acidic amino acid residue; d¹¹ is an aminoacid residue; d¹² is a neutral hydrophobic, neutral polar, or basicamino acid residue; d¹⁵ is absent, or a neutral polar, acidic, or abasic amino acid residue; d¹⁶ is absent, or a neutral polar, acidic, ora basic amino acid residue; d¹⁷ is absent, or a neutral hydrophobic, orneutral polar amino acid residue; d¹⁸ is absent, or a neutralhydrophobic, or neutral polar amino acid residue; d¹⁹ is absent, or aneutral hydrophobic, neutral polar, or basic amino acid residue; d²⁰ isabsent, or an amino acid residue; d²¹ is absent, or a neutral polar,acidic, or a basic amino acid residue; d²² is absent, or a neutralhydrophobic, neutral polar, or basic amino acid residue. Physiologicallyacceptable salts thereof are also suitable.

In another preferred embodiment, d¹ is T, S, Q, R, or H; d² is T, Q, N,or K; d³ is F; d⁴ is M, Q, E, or K; d⁶ is L or M; d⁸ is D or E; d¹⁰ isE; d¹¹ is Q or E; d¹² is T or R; d¹⁵ Y, D, E, or K; d¹⁶ is Q; d¹⁷ is Wor F; d¹⁸ is L, I, M, or T; d¹⁹ is L, F, or Y; d²⁰ is Q, D, or E; d²¹ isabsent, Q, or H; d²² is absent, A, L, G, S, or R. Physiologicallyacceptable salts thereof are also suitable.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein the peptide or peptibody comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO: 6,and SEQ ID NO: 119 to SEQ ID NO: 142, inclusive, and physiologicallyacceptable salts thereof.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein said peptide or peptibody comprises an amino acidsequence of the formula:

RPe³e⁴e⁵e⁶e⁷ G (SEQ ID NO: 73)

wherein: e³ is a neutral polar amino acid residue; e⁴ is an acidic aminoacid residue; e⁵ is a neutral polar or an acidic amino acid residue; e⁶is a neutral hydrophobic amino acid residue; e⁷ is a neutral hydrophobicamino acid residue; and physiologically acceptable salts thereof. In apreferred embodiment, e³ is Y or C. In another preferred embodiment, e⁴is D or E. In another preferred embodiment, e⁶ is I or M.Physiologically acceptable salts thereof are also suitable.

In another embodiment, the peptide or peptibody comprises an amino acidsequence of the formula:

(SEQ ID NO: 74) f¹f²f³f⁴ RPf⁷f⁸f⁹f¹⁰f¹¹ Gf¹³f¹⁴f¹⁵f¹⁶f¹⁷f¹⁸f¹⁹f²⁰

wherein: f¹ is a neutral hydrophobic or neutral polar amino acidresidue; f² is a neutral hydrophobic or neutral polar amino acidresidue; f³ is a neutral polar or acidic amino acid residue; f⁴ is aneutral hydrophobic or neutral polar amino acid residue; f⁷ is a neutralpolar amino acid residue; f⁸ is an acidic amino acid residue; f⁹ is aneutral polar or acidic amino acid residue; f¹⁰ is a neutral hydrophobicamino acid residue; f¹¹ is a neutral hydrophobic amino acid residue; f¹³is a neutral hydrophobic or neutral polar amino acid residue; f¹⁴ is aneutral hydrophobic or neutral polar amino acid residue; f¹⁵ is aneutral polar amino acid residue; f¹⁶ is a neutral polar amino acidresidue; f¹⁷ is a neutral polar or acidic amino acid residue; f¹⁸ is aneutral hydrophobic or basic amino acid residue; f¹⁹ is a neutralhydrophobic or neutral polar amino acid residue; and f²⁰ is a neutralhydrophobic or neutral polar amino acid residue; and physiologicallyacceptable salts thereof. In a preferred embodiment, f¹ is S, A, or G;f² is G, Q, or P; f³ is Q, G, or D; f⁴ is L, M, or Q; f⁷ is C or Y; f⁸is E or D; f⁹ is E, G, or D; f¹⁰ is I or M; f¹¹ is For L; f¹³ is C or W;f¹⁴ is G or P; f¹⁵ T or N; f¹⁶ is Q, Y, or K; f¹⁷ is N, D, or Q; f¹⁸ isL, V, W, or R; f¹⁹ is A, Q, Y, or I; and f²⁰ is L, A, G, or V.Physiologically acceptable salts thereof are also suitable.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein the peptide or peptibody comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO: 3,and SEQ ID NO: 143 to SEQ ID NO: 148, inclusive, and physiologicallyacceptable salts thereof.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein said peptide or peptibody comprises an amino acidsequence of the formula:

Cg² Gg⁴g⁵ DPFTg¹⁰ GCg¹³ (SEQ ID NO: 75)

wherein: g² is an acidic amino acid residue; g⁴ is a neutral hydrophobicamino acid residue; g⁵ is E, D, or Q; g¹⁰ is a neutral hydrophobic orneutral polar amino acid residue; g¹³ is an acidic residue; andphysiologically acceptable salts thereof.

In another embodiment, the peptide or peptibody comprises an amino acidsequence of the formula:

(SEQ ID NO: 158) h¹h²h³h⁴ Ch⁶ Gh⁸h⁹ DPFTh¹⁴ GCh¹⁷h¹⁸h¹⁹h²⁰

wherein: h¹ is absent or a neutral hydrophobic, neutral polar, or abasic amino acid residue; h² is a neutral hydrophobic or neutral polaramino acid residue; h³ is an acidic amino acid residue; h⁴ is a neutralhydrophobic or neutral polar amino acid residue; h⁶ is an acidic aminoacid residue; h⁸ is a neutral hydrophobic amino acid residue; h⁹ is E,D, or Q; h¹⁴ is a neutral hydrophobic or neutral polar amino acidresidue; h¹⁷ is an acidic amino acid residue; h¹⁸ is a neutralhydrophobic, neutral polar, or a basic amino acid residue; h¹⁹ is aneutral hydrophobic or neutral polar amino acid residue; and h²⁰ isabsent or an amino acid residue. Physiologically acceptable saltsthereof are also suitable. In another preferred embodiment, h¹ isabsent, or A, L, M, G, K, or H; h² is L, F, or Q; h³ is D or E; h⁴ is Wor Y; h⁶ is D or E; h⁸ is V or M; h¹⁴ is F or Q; h¹⁷ is D or E; h¹⁸ isM, Y, N, or K; h¹⁹ is L or Q; and h²⁰ is absent or M, T, G, S, D, K, orR. Physiologically acceptable salts thereof are also suitable.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a peptide or peptibody capable ofbinding Ang-2, wherein the peptide or peptibody comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO: 5,and SEQ ID NO: 149 to SEQ ID NO: 157, inclusive, and physiologicallyacceptable salts thereof.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a composition of matter of theformula:(X¹)_(a)-F¹-(X²)_(b)

-   -   and multimers thereof, wherein:        -   F¹ is a vehicle;        -   X¹ and X² are each independently selected from            -   -(L¹)_(c)-P¹;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and            -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴;    -   wherein a, b, c, d, e, and f are each independently 0 or 1,        provided that at least one of a and b is 1; wherein L¹, L², L³,        and L⁴ are each independently linkers; and wherein one or more        of P¹, P², P³, and P⁴ each independently comprise a peptide        having an amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 66,        SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69; and        physiologically acceptable salts thereof.

The invention further relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a composition of matter of theformula:(X¹)_(a)-F¹-(X²)_(b)

-   -   and multimers thereof, wherein:        -   F¹ is a vehicle;        -   X¹ and X² are each independently selected from            -   -(L¹)_(c)-P¹;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and            -   -(L¹)_(c)-P¹-(L²)_(d)-P² -(L³)_(e)-P³-(L⁴)_(f)-P⁴;    -   wherein a, b, c, d, e, and f are each independently 0 or 1,        provided that at least one of a and b is 1; wherein L¹, L², L³,        and L⁴ are each independently linkers; and wherein one or more        of P¹, P², P³, and P⁴ each independently comprise a peptide        having an amino acid sequence of SEQ ID NO: 71, and 72; and        physiologically acceptable salts thereof.

The invention also relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a composition of matter of theformula:(X¹)_(a)-F¹-(X²)_(b)

-   -   and multimers thereof, wherein:        -   F¹ is a vehicle;        -   X¹ and X² are each independently selected from            -   -(L¹)_(c)-P¹;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and            -   -(L¹)_(c)-P¹-(L²)_(d)-P² -(L³)_(e)-P³-(L⁴)_(f)-P⁴;    -   wherein a, b, c, d, e, and f are each independently 0 or 1,        provided that at least one of a and b is 1; wherein L¹, L², L³,        and L⁴ are each independently linkers; and wherein one or more        of P¹, P², P³, and P⁴ each independently comprise a peptide        having an amino acid sequence of SEQ ID NO: 73, and SEQ ID NO:        74; and physiologically acceptable salts thereof.

The invention also relates to a method for treating an inflammatorydisease which comprises administering to a patient in need thereof atherapeutically effective amount of a composition of matter of theformula:(X¹)_(a)-F¹-(X²)_(b)

-   -   and multimers thereof, wherein:        -   F¹ is a vehicle;        -   X¹ and X² are each independently selected from            -   -(L¹)_(c)-P¹;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²;            -   -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and            -   -(L¹)_(c)-P¹-(L²)_(d)-P² -(L³)_(e)-P³-(L⁴)_(f)-P⁴;    -   wherein a, b, c, d, e, and f are each independently 0 or 1,        provided that at least one of a and b is 1; wherein L¹, L², L³,        and L⁴ are each independently linkers; and wherein one or more        of P¹, P², P³, and P⁴ each independently comprise a peptide        having an amino acid sequence of SEQ ID NO: 75, and SEQ ID NO:        158; and physiologically acceptable salts thereof.

It will be appreciated that the invention also relates to methods fortreating inflammatory disease using the specific binding agents (such aspeptides, peptibodies, or compositions of matter) described herein, andfurther comprising administering at least one anti-inflammatory agent.In one preferred embodiment, the anti-inflammatory agent may comprise atleast one of a DMARD, SAARD, and NSAID. In another preferred embodiment,the anti-inflammatory agent may comprise at least one of methotrexate, aTNF inhibitor, a IL-1 inhibitor, a TACE inhibitor, a COX-2 inhibitor,and a P-38 inhibitor. In still another preferred embodiment, the TNFinhibitor comprises at least one of etanercept, adalimumab, pegsunerceptsTNF-R1, onercept, and infliximab. In yet another preferred embodiment,the IL-1 inhibitor may be at least one of anakinra, IL-1 TRAP, IL-1antibody, and soluble IL-1 receptor.

As described herein, it will be appreciated that the administration canbe concurrent administration, or non-concurrent administration.

In yet another preferred embodiment, the invention relates to a methodfor treating an inflammatory disease which comprises administering to apatient in need thereof a therapeutically effective amount of a peptideor peptibody capable of binding Ang-2, wherein said peptide or peptibodycomprises the amino acid sequence set forth in SEQ ID NO: 4 or SEQ IDNO: 25. In still another preferred embodiment, the invention relates toa method for treating an inflammatory disease which comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a peptide or peptibody capable of binding Ang-2, wherein saidpeptide or peptibody comprises the amino acid sequence set forth in SEQID NO: 124, 126, or 137. These methods may also further compriseadministering at least one anti-inflammatory agent. The administrationcan be concurrent administration, or non-concurrent administration.

Other embodiments of this invention will be readily apparent from thedisclosure provided herewith.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph of tumor volume (y-axis) versus time (x-axis) inA431 tumor bearing mice treated with peptibody TN8-Con4-C of the presentinvention, or with phosphate buffered saline (PBS). Details aredescribed in the Examples.

FIG. 2 depicts a graph of peptibody concentration (y-axis) versus timepost-dose (x-axis) in wildtype mice treated with a 50 μg dose of either2xCon4-C, L1-7-N, or L1-21-N peptibody. Details are described in theExamples.

FIG. 3 depicts a graph of tumor volume (y-axis) versus time (x-axis) inA431 tumor bearing mice treated with peptibody 2xCon4-C according to thepresent invention, or with phosphate buffered saline (PBS) or controlpeptibody. Details are described in the Examples.

FIG. 4 depicts a graph representing in vitro growth of cultured A431cells treated with peptibody Con4-C according to the present invention,control peptibody, or untreated. Details are described in the Examples.

FIG. 5 depicts a graph of tumor volume (y-axis) versus time (x-axis) inColo205 tumor cells treated with peptibody Con4-C, peptibody L1-7-N,peptibody L1-21-N, or peptibody 2xCon4-C according to the presentinvention, or with phosphate buffered saline (PBS), anti-Ang-2 antibody(Ab536), or Fc. Details are described in the Examples.

FIG. 6 depicts a graph of tumor volume (y-axis) versus time (x-axis) inColo205 xenograft tumor bearing mice treated with varying doses ofpeptibody 2xCon4-C according to the present invention, or with phosphatebuffered saline (PBS) or Fc. Details are described in the Examples.

FIG. 7 depicts a graph of tumor volume (y-axis) versus time (x-axis) inColo205 xenograft tumor bearing mice treated with peptibody 2xCon4-Caccording to the present invention, or with control peptibodies. FIG. 7also depicts a graph of CD31 stained area/total tumor area for thesepeptibodies. Details are described in the Examples.

FIG. 8 depicts a graph of tumor volume (y-axis) versus time (x-axis) inColo205 xenograft tumor bearing mice treated with peptibody 2xCon4-Caccording to the present invention, or with phosphate buffered saline(PBS) or control peptibody. Details are described in the Examples. Thisgraph shows that anti-Ang-2 peptibodies are capable of inhibitingColo205 tumor growth irrespective of when dosing begins.

FIG. 9 depicts a summary of complete response (CR) rates obtained infemale nude mice using antibody Ab536 or with peptibody 2xCon4-C, inboth the A431 and Colo-205 xenograft models. Details are described inthe Examples.

FIG. 10A depicts a graph of tumor volume (y-axis) versus time (x-axis)in Colo205 xenograft tumor bearing mice treated with peptibody 2xCon4-Caccording to the present invention, or a combination of 2xCon4-C andtaxotere, or with phosphate buffered saline (PBS), or with PBS plustaxotere. Details are described in the Examples.

FIG. 10B depicts a graph of tumor volume (y-axis) versus time (x-axis)in Colo205 xenograft tumor bearing mice treated with peptibody 2xCon4-Caccording to the present invention, or a combination of 2xCon4-C and5-FU, or with phosphate buffered saline (PBS), or with PBS plus 5-FU.Details are described in the Examples.

FIG. 11A depicts a graph of paw swelling levels (AUC±SE) in anadjuvant-induced arthritis model in rats treated with peptibody 2xCon4-Caccording to the present invention, or with phosphate buffered saline(PBS), or with control peptibody, or normal or arthritis controls.Details are described in the Examples.

FIG. 11B depicts a graph of paw bone mineral density (BMD) in anadjuvant-induced arthritis model in rats treated with peptibody 2xCon4-Caccording to the present invention, or with phosphate buffered saline(PBS), or with control peptibody, or normal or arthritis controls.Details are described in the Examples.

FIG. 11C depicts a graph of change in body weight in an adjuvant-inducedarthritis model in rats treated with peptibody 2xCon4-C according to thepresent invention, or with phosphate buffered saline (PBS), or withcontrol peptibody, or normal or arthritis controls. Details aredescribed in the Examples.

FIG. 12 depicts two graphs depicting inhibition of VEGF-induced cornealangiogenesis in rats. The first graph depicts number of blood vesselsmeasured in rats treated with bovine serum albumin (BSA), VEGF plusphosphate buffered saline (PBS), or VEGF plus peptibody Con4-C of theinvention. The second graph depicts blood vessel area (mm²) in ratstreated with BSA, VEGF plus phosphate buffered saline (PBS), or VEGFplus peptibody Con4-C of the invention. Details are described in theExamples.

FIGS. 13A, 13B, and 13C depict epitope mapping data (O.D. 370) forfull-length human Ang-2 (hAng-2), to the N-terminus of hAng-2, and tothe C-terminus of hAng-2, respectively, for peptibodies TN8-Con4-C,L1-7-N, and 12-9-3-C according to the invention, as well as for controlpeptibody, Tie2-Fc, C2B8, or 5B12. Details are described in theExamples.

FIG. 14 depicts binding affinity (K_(D))of the 2xCon4-C peptibodyaccording to the invention, using the Sapidyne KinExA assay. Details aredescribed in the Examples.

FIG. 15 depicts the effects of peptibody 2xCon4(C) and theanti-inflammatory agent, PEG sTNF-R1, on Paw Swelling in Rat AdjuvantArthritis. Details are described in the Examples.

FIG. 16 also depicts the effects of peptibody 2xCon4(C) and theanti-inflammatory agent, PEG sTNF-R1, on AUC paw swelling in RatAdjuvant Arthritis. Details are described in the Examples.

FIG. 17 depicts the effects of peptibody 2xCon4(C) and theanti-inflammatory agent, PEG sTNF-R 1, on Body Weight in Rat AdjuvantArthritis. Details are described in the Examples.

DETAILED DESCRIPTION OF INVENTION

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed.

Standard techniques may be used for recombinant DNA molecule, protein,and antibody production, as well as for tissue culture and celltransformation. Enzymatic reactions and purification techniques aretypically performed according to the manufacturer's specifications or ascommonly accomplished in the art using conventional procedures such asthose set forth in Sambrook et al. (Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.[1989]), or as described herein. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques may be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

Definitions

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances.

The term “Ang-2” refers to the polypeptide set forth in FIG. 6 of U.S.Pat. No. 6,166,185 (“Tie-2 ligand-2”) or fragments thereof as well asrelated polypeptides which include allelic variants, splice variants,derivatives, substitution, deletions, and/or insertion variants, fusionpeptides and polypeptides, and interspecies homologs. The Ang-2polypeptide may or may not include additional terminal residues, e.g.,leader sequences, targeting sequences, amino terminal methionine, aminoterminal methionine and lysine residues, and/or tag or fusion proteinssequences, depending on the manner in which it is prepared.

The term “biologically active” when used in relation to Ang-2 or anAng-2 specific binding agent refers to a peptide or polypeptide havingat least one activity characteristic of Ang-2 or of an Ang-2 specificbinding agent. A specific binding agent of Ang-2 may have agonist,antagonist, or neutralizing or blocking activity with respect to atleast one biological activity of Ang-2.

The term “specific binding agent” refers to a molecule, preferably aproteinaceous molecule, that specifically binds Ang-2, and variants andderivatives thereof, as defined herein. A specific binding agent may bea protein, peptide, nucleic acid, carbohydrate, lipid, or smallmolecular weight compound which binds preferentially to Ang-2. In apreferred embodiment, the specific binding agent according to thepresent invention is a peptide or a peptibody, as well as fragments,variants or derivatives thereof, either alone or in combination withother amino acid sequences, provided by known techniques. Suchtechniques include, but are not limited to enzymatic cleavage, chemicalcleavage, peptide synthesis or recombinant techniques. The anti-Ang-2specific binding agents of the present invention are capable of bindingportions of Ang-2 that modulate, e.g., inhibit or promote, thebiological activity of Ang-2 and/or other Ang-2-associated activities.

The term “variants,” as used herein, include those peptides andpolypeptides wherein amino acid residues are inserted into, deleted fromand/or substituted into the naturally occurring (or at least a known)amino acid sequence for the binding agent. Variants of the inventioninclude fusion proteins as described below.

“Derivatives” include those binding agents that have been chemicallymodified in some manner distinct from insertion, deletion, orsubstitution variants.

“Specifically binds Ang-2” refers to the ability of a specific bindingagent (such as a peptibody, or peptide portion thereof) of the presentinvention to recognize and bind mature, full-length or partial-lengthhuman Ang-2 polypeptide, or an ortholog thereof, such that its affinity(as determined by, e.g., Affinity ELISA or BIAcore assays as describedherein) or its neutralization capability (as determined by e.g.,Neutralization ELISA assays described herein, or similar assays) is atleast 10 times as great, but optionally 50 times as great, 100, 250 or500 times as great, or even at least 1000 times as great as the affinityor neutralization capability of the same for any other angiopoietin orother peptide or polypeptide, wherein the peptide portion of thepeptibody is first fused to a human Fc moiety for evaluation in suchassay.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a specific binding agent, e.g., apeptibody, at one or more of the binding agent's antigen bindingregions. Epitopes usually consist of chemically active surface groupingsof molecules, such as for example, amino acids or carbohydrate sidechains, and have specific three-dimensional structural characteristicsas well as specific charge characteristics. Epitopes as used herein maybe contiguous or non-contiguous.

The term “inhibiting and/or neutralizing epitope” is an epitope, whichwhen bound by a specific binding agent such as a peptibody, results inthe loss of (or at least the decrease in) biological activity of themolecule, cell, or organism containing such epitope, in vivo, in vitro,or in situ. In the context of the present invention, the neutralizingepitope is located on or is associated with a biologically active regionof Ang-2. Alternatively, the term “activating epitope” is an epitope,which when bound by a specific binding agent of the invention, such asan antibody, results in activation, or at least maintenance of abiologically active conformation, of Ang-2.

The term “peptibody fragment” refers to a peptide or polypeptide whichcomprises less than a complete, intact peptibody.

The term “naturally occurring” when used in connection with biologicalmaterials such as nucleic acid molecules, polypeptides, host cells, andthe like, refers to those which are found in nature and not modified bya human being.

The term “isolated” when used in relation to Ang-2 or to a specificbinding agent of Ang-2 refers to a compound that is free from at leastone contaminating polypeptide or compound that is found in its naturalenvironment, and preferably substantially free from any othercontaminating mammalian polypeptides that would interfere with itstherapeutic or diagnostic use.

The term “mature” when used in relation to Ang-2 peptibody or a fragmentthereof, or to any other proteinaceous specific binding agent of Ang-2refers to a peptide or a polypeptide lacking a leader or signalsequence. When a binding agent of the invention is expressed, forexample, in a prokaryotic host cell, the “mature” peptide or polypeptidemay also include additional amino acid residues (but still lack a leadersequence) such as an amino terminal methionine, or one or moremethionine and lysine residues. A peptide or polypeptide produced inthis manner may be utilized with or without these additional amino acidresidues having been removed.

The terms “effective amount” and “therapeutically effective amount” whenused in relation to a specific binding agent of Ang-2 refers to anamount of a specific binding agent that is useful or necessary tosupport an observable change in the level of one or more biologicalactivities of Ang-2. The change may be either an increase or decrease inthe level of Ang-2 activity. Preferably, the change is a decrease inAng-2 activity.

The term “peptibody” refers to a molecule comprising an antibody Fcdomain attached to at least one peptide. The production of peptibodiesis generally described in PCT publication WO 00/24782, published May 4,2000.

The term “variants,” as used herein, include those molecules such aspeptides or peptide-vehicle combinations such as peptibodies of thepresent invention wherein amino acid residues are inserted into, deletedfrom and/or substituted into amino acid sequence for such molecules.Variants having one or more amino acids inserted include fusion proteinsas described below.

“Derivatives” include those peptides and/or peptide-vehicle combinationssuch as peptibodies that have been chemically modified in some mannerdistinct from insertion, deletion, or substitution variants.

The term “fragment” refers to a peptide or peptide-vehicle combinationthat comprises less than the full-length amino acid sequence of suchpeptides and/or peptide-vehicle combinations. Such a fragment may arise,for example, from a truncation at the amino terminus, a truncation atthe carboxy-terminus, and/or an internal deletion of a residue(s) fromthe amino acid sequence of the peptide or peptide-vehicle combination.Fragments may result from alternative RNA splicing or from in vivo or invitro protease activity. Such fragments may also be constructed bychemical peptide synthesis methods, or by modifying a polynucleotideencoding a peptide, peptide-vehicle combination, or an Fc portion and/orpeptide portion of a peptibody.

The term “Fc” refers to one type of vehicle of the present invention,and comprises the sequence of a non-antigen-binding fragment of anantibody resulting from the proteolytic digestion of a whole antibody,whether in monomeric or multimeric form. The source of the Fc in thepresent invention is preferably fully human Fc, and may be any of theimmunoglobulins, although IgG1 and IgG2 are preferred. However, Fcmolecules that are partially human, or obtained from non-human speciesare also included herein. Fc's are made up of monomeric polypeptidesthat may be linked into dimeric or multimeric forms by covalent (i.e.,disulfide bonds) and non-covalent association. The number ofintermolecular disulfide bonds between monomeric subunits of native Fcmolecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) orsubclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a nativeFc is a disulfide-bonded dimer resulting from papain digestion of an IgG[see Ellison et al. (1982), Nucl. Acids. Res. 10: 4071-9]. The term“native Fc” as used herein is generic to the monomeric, dimeric, andmultimeric forms.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently.

The term “vehicle” refers to a molecule that prevents degradation and/orincreases half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of a therapeutic protein. Exemplaryvehicles include an Fc domain as well as a linear polymer (e.g.,polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chainpolymer (See, for example, U.S. Pat. No. 4,289,872 to Denkenwalter etal., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul.20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); alipid; a cholesterol group (such as a steroid); a carbohydrate oroligosaccharide; or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor. Vehicles are further describedhereinafter.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of about 3 to about 75 aminoacids, with molecules of about 5 to 50 amino acids preferred, 8 to 40more preferred, and those of about 10 to 25 amino acids most preferred.Peptides may be naturally occurring or artificial (i.e., non-naturallyoccurring) amino acid sequences. Exemplary peptides may be generated byany of the methods set forth herein, such as carried in a peptidelibrary (e.g., a phage display library), generated by chemicalsynthesis, derived by digestion of proteins, or generated usingrecombinant DNA techniques.

The term “pharmacologically active” means, that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer, autoimmune disorders, etc.).

The terms “antagonist peptide” or “inhibitor peptide” refer to a peptidethat blocks or in some way interferes with the biological activity ofthe associated protein of interest, or has biological activitycomparable to a known antagonist or inhibitor of the associated proteinof interest. Thus, the term “Ang-2-antagonist peptide” comprisespeptides that can be identified or derived as having Ang-2-antagonisticcharacteristics.

Additionally, physiologically acceptable salts of the compounds of thisinvention are also encompassed herein. By “physiologically acceptablesalts” is meant any salts that are known or later discovered to bepharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate, mesylate, andphosphate.

Peptibodies

One aspect of the present invention relates to development of Ang-2peptibodies. The interaction of a protein ligand with its receptor oftentakes place at a relatively large interface. However, as demonstratedfor human growth hormone and its receptor, only a few key residues atthe interface contribute to most of the binding energy. Clackson et al.,Science 267: 383-6 (1995). The bulk. of the protein ligand merelydisplays the binding epitopes in the right topology or serves functionsunrelated to binding. Thus, molecules of only “peptide” length(generally 2 to 40 amino acids) can bind to the receptor protein of agiven large protein ligand. Such peptides may mimic the bioactivity ofthe large protein ligand (“peptide agonists”) or, through competitivebinding, inhibit the bioactivity of the large protein ligand (“peptideantagonists”).

Phage display technology has emerged as a powerful method in identifyingsuch peptide agonists and antagonists. See, for example, Scott et al.Science 249: 386 (1990); Devlin et al., Science 249: 404 (1990); U.S.Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731,issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996;U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665,issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16,1998 (each of which is incorporated by reference). In peptide phagedisplay libraries, random peptide sequences can be displayed by fusionwith coat proteins of filamentous phage. The displayed peptides can beaffinity-eluted against an antibody-immobilized extracellular domain ofa receptor, if desired. The retained phage may be enriched by successiverounds of affinity purification and repropagation. The best bindingpeptides may be sequenced to identify key residues within one or morestructurally related families of peptides. See, e.g., Cwirla et al.,Science 276: 1696-9 (1997), in which two distinct families wereidentified. The peptide sequences may also suggest which residues may besafely replaced by alanine scanning or by mutagenesis at the DNA level.Mutagenesis libraries may be created and screened to further optimizethe sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol.Struct. 26: 401-24 (1997).

Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed. See, e.g.,Takasaki et al., Nature Biotech 15: 1266-70 (1997). These analyticalmethods may also be used to investigate the interaction between areceptor protein and peptides selected by phage display, which maysuggest further modification of the peptides to increase bindingaffinity.

Other methods compete with phage display in peptide research. A peptidelibrary can be fused to the carboxyl terminus of the lac repressor andexpressed in E. coli. Another E. coli-based method allows display on thecell's outer membrane by fusion with a peptidoglycan-associatedlipoprotein (PAL). Hereinafter, these and related methods arecollectively referred to as “E. coli display.” In another method,translation of random RNA is halted prior to ribosome release, resultingin a library of polypeptides with their associated RNA still attached.Hereinafter, this and related methods are collectively referred to as“ribosome display.” Other methods employ chemical linkage of peptides toRNA. See, for example, Roberts and Szostak, Proc Natl Acad Sci USA, 94:12297-303 (1997). Hereinafter, this and related methods are collectivelyreferred to as “RNA-peptide screening.” Chemically derived peptidelibraries have been developed in which peptides are immobilized onstable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.Hereinafter, these and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells and Lowman, Curr. Opin. Biotechnol., 3:355-62 (1992).

Conceptually, one may discover peptide mimetics of any protein usingphage display and the other methods mentioned above. These methods havebeen used for epitope mapping, for identification of critical aminoacids in protein-protein interactions, and as leads for the discovery ofnew therapeutic agents. See, e.g., Cortese et al., Curr. Opin. Biotech.7: 616-21 (1996). Peptide libraries are now being used most often inimmunological studies, such as epitope mapping. See Kreeger, TheScientist 10 (13):19-20(1996).

Peptides identified by phage display library screening have beenregarded as “leads” in development of therapeutic agents rather than astherapeutic agents themselves. Like other proteins and peptides, theywould likely be rapidly removed in vivo either by renal filtration, bycellular clearance mechanisms in the reticuloendothelial system, or byproteolytic degradation [Francis, (supra)]. As a result, the artpresently uses peptides to validate drug targets or as scaffolds fordesign of organic compounds that might not have been as easily or asquickly identified through chemical library screening [Lowman, (supra);Kay et al., (supra)]. The art would benefit from a process by which suchpeptides could more readily yield therapeutic agents againstangiogenesis.

Structure of Peptibodies

In the compositions of matter prepared in accordance with thisinvention, the peptide may be attached to a vehicle through thepeptide's N-terminus or C-terminus. Thus, vehicle-peptide molecules ofthis invention may be described by the following five formulae andmultimers thereof:

(X₁)_(a)-F₁-(X₂)_(b) (FORMULA I) X₁-F₁ (FORMULA II) F₁-X₂ (FORMULA III)F₁-(L₁)_(c)-P₁ (FORMULA IV) F₁-(L₁)_(c)-P₁-(L₂)_(d)-P₂ (FORMULA V)wherein:

F₁ is a vehicle (preferably an Fc domain);

X₁ and X₂ are each independently selected from -(L₁)_(c)-P₁,-(L₁)_(c)-P₁-(L₂)_(d)-P₂, -(L₁)_(c)-P₁-(L₂)_(d)-P₂ -(L₃)_(e)-P₃, and-L₁)_(c)-P₁-(L₂)_(d)-P₂ -(L₃)_(e)-P₃-(L₄)_(f)-P₄

P₁, P₂, P₃, and P₄ are each independently sequences of pharmacologicallyactive peptides as described herein;

L₁, L₂, L₃, and L₄ are each independently linkers; and

“a”, “b”, “c”, “d”, “e”, and “f” are each independently 0 or 1, providedthat at least one of “a” and “b” is 1.

Peptides

The present invention contemplates peptides that selectively bind orspecifically bind to Ang-2. Any number of such peptides may be used inconjunction with the present invention. Phage display, in particular, isuseful in generating peptides for use in the present invention as hasbeen shown that affinity selection from libraries of random peptides canbe used to identify peptide ligands for any site of any gene product.Dedman et al., J. Biol. Chem. 268: 23025-30 (1993).

The peptides in this invention may be prepared by any of the methodsdisclosed in the art. Single letter amino acid abbreviations are used.The “X” in any sequence (and throughout this specification, unlessspecified otherwise in a particular instance) means that any of the 20naturally occurring amino acid residues, or any non-naturally occurringamino acids (described below under “Variants”), may be present. Any ofthese peptides may be linked in tandem (i.e., sequentially), with orwithout linkers, and tandem-linked examples are provided in the table.Linkers are listed as “L” and may be any of the linkers describedherein. Tandem repeats and linkers are shown separated by dashes forclarity. Any peptide containing a cysteinyl residue may be cross-linkedwith another Cys-containing peptide, either or both of which may belinked to a vehicle. Any peptide having more than one Cys residue mayform an intrapeptide disulfide bond, as well. Any of these peptides maybe derivatized as described herein. For derivatives in which thecarboxyl terminus may be capped with an amino group, the capping aminogroup is —NH₂. For derivatives in which amino acid residues aresubstituted by moieties other than amino acid residues, thesubstitutions are denoted by S, which signifies any of the moietiesdescribed in Bhatnagar et al., J. Med. Chem. 39: 3814-9 (1996), andCuthbertson et al., J. Med. Chem. 40: 2876-82 (1997), which areincorporated by reference. All peptides are linked through peptide bondsunless otherwise noted.

Vehicles

In one embodiment, this invention provides for at least one peptide tobe attached to at least one vehicle (F₁, F₂) through the N-terminus,C-terminus or a side chain of one of the amino acid residues of thepeptide(s). Multiple vehicles may also be used; e.g., Fc's at eachterminus or an Fc at a terminus and a PEG group at the other terminus ora side chain.

An Fc domain is one preferred vehicle. The Fc domain may be fused to theN or C termini of the peptides or at both the N and C termini.

As noted above, Fc variants are suitable vehicles within the scope ofthis invention. A native Fc may be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained. See, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removalmay avoid reaction with other cysteine-containing proteins present inthe host cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus may betruncated or cysteine residues may be deleted or substituted with otheramino acids (e.g., alanyl, seryl). Even when cysteine residues areremoved, the single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selectedhost cell. For example, one may remove the PA sequence near theN-terminus of a typical native Fc, which may be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One may alsoadd an N-terminal methionyl residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli.

3. A portion of the N-terminus of a native Fc is removed to preventN-terminal heterogeneity when expressed in a selected host cell. Forthis purpose, one may delete any of the first 20 amino acid residues atthe N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) may confer cytolytic response.Such residues may be deleted or substituted with unglycosylated residues(e.g., alanine).

5. Sites involved in interaction with complement, such as the Clqbinding site, are removed. For example, one may delete or substitute theEKK sequence of human IgG1. Complement recruitment may not beadvantageous for the molecules of this invention and so may be avoidedwith such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than asalvage receptor. A native Fc may have sites for interaction withcertain white blood cells that are not required for the fusion moleculesof the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art. See, forexample, Molec. Immunol. 29 (5):633-9 (1992) with regard to ADCC sitesin IgG1. These sites, as well, are not required for the fusion moleculesof the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the nativeFc may be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

An alternative vehicle would be a protein, polypeptide, peptide,antibody, antibody fragment, or small molecule (e.g., a peptidomimeticcompound) capable of binding to a salvage receptor. For example, onecould use as a vehicle a polypeptide as described in U.S. Pat. No.5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also beselected by phage display for binding to the FcRn salvage receptor. Suchsalvage receptor-binding compounds are also included within the meaningof “vehicle” and are within the scope of this invention. Such vehiclesshould be selected for increased half-life (e.g., by avoiding sequencesrecognized by proteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

As noted above, polymer vehicles may also be used for F₁, and F₂.Various means for attaching chemical moieties useful as vehicles arecurrently available, see, e.g., Patent Cooperation Treaty (“PCT”)International Publication No. WO 96/11953, entitled “N-TerminallyChemically Modified Protein Compositions and Methods,” hereinincorporated by reference in its entirety. This PCT publicationdiscloses, among other things, the selective attachment of water solublepolymers to the N-terminus of proteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be linear or branched.The average molecular weight of the PEG will preferably range from about2 kiloDalton (“kDa”) to about 100 kDa, more preferably from about 5 kDato about 50 kDa, most preferably from about 5 kDa to about 10 kDa. ThePEG groups will generally be attached to the compounds of the inventionvia acylation or reductive alkylation through a reactive group on thePEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the inventive compound (e.g., an aldehyde, amino, orester group).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis as known in the art. The peptides are“preactivated” with an appropriate functional group at a specific site.The precursors are purified and fully characterized prior to reactingwith the PEG moiety. Ligation of the peptide with PEG usually takesplace in aqueous phase and can be easily monitored by reverse phaseanalytical HPLC. The PEGylated peptides can be easily purified bypreparative HPLC and characterized by analytical HPLC, amino acidanalysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by al-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kDa to about 70 kDa. Dextran is a suitable water-solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference. Dextran of about 1 kDa to about 20 kDa is preferred whendextran is used as a vehicle in accordance with the present invention.

Linkers

Any “linker” group is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer. The linker ispreferably made up of amino acids linked together by peptide bonds.Thus, in preferred embodiments, the linker is made up of from 1 to 20amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring anino acids. One or more ofthese amino acids may be glycosylated, as is well understood by those inthe art. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines (particularly (Gly)₅, (Gly)₈),poly(Gly-Ala), and polyalanines. Combinations of Gly and Ala are alsopreferred as is the linker referred to herein as K1 and having an aminoacid sequence set forth in the Examples herein.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)s—C(O)—, wherein s=2-20 can be. used. These alkyl linkersmay further be substituted by any non-sterically hindering group such aslower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂,phenyl, etc. An exemplary non-peptide linker is a PEG linker, and has amolecular weight of 100 to 5000 kDa, preferably 100 to 500 kDa. Thepeptide linkers may be altered to form derivatives in the same manner asdescribed above.

Variants and Derivatives

Variants and derivatives of the specific binding agents are includedwithin the scope of the present invention. Included within variants areinsertional, deletional, and substitutional variants. It is understoodthat a particular specific binding agent of the present invention maycontain one, two or all three types of variants. Insertional andsubstitutional variants may contain natural amino acids, unconventionalamino acids (as set forth below), or both.

In one example, insertional variants are provided wherein one or moreamino acid residues, either naturally occurring or unconventional aminoacids, supplement a peptide or a peptibody amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the peptibody amino acidsequence. Insertional variants with additional residues at either orboth termini can include, for example, fusion proteins and proteinsincluding amino acid tags or labels. Insertion variants include peptidesand peptibodies wherein one or more amino acid residues are added to thepeptide or peptibody amino acid sequence, or fragment thereof.

Variant products of the invention also include mature peptides andpeptibodies wherein leader or signal sequences are removed, and theresulting proteins having additional amino terminal residues, whichamino acids may be natural or non-natural. Specific binding agents (suchas peptibodies) with an additional methionyl residue at amino acidposition −1 (Met⁻¹-peptibody) are contemplated, as are specific bindingagents with additional methionine and lysine residues at positions −2and −1 (Met⁻²-Lys⁻¹). Variants having additional Met, Met-Lys, Lysresidues (or one or more basic residues, in general) are particularlyuseful for enhanced recombinant protein production in bacterial hostcells.

The invention also embraces specific binding agent variants havingadditional amino acid residues that arise from use of specificexpression systems. For example, use of commercially available vectorsthat express a desired polypeptide as part of glutathione-S-transferase(GST) fusion product provides the desired polypeptide having anadditional glycine residue at amino acid position −1 after cleavage ofthe GST component from the desired polypeptide. Variants which resultfrom expression in other vector systems are also contemplated, includingthose wherein poly-histidine tags are incorporated into the amino acidsequence, generally at the carboxy and/or amino terminus of thesequence.

Insertional variants also include fusion proteins wherein the aminoand/or carboxy termini of the peptide or peptibody is fused to anotherpolypeptide, a fragment thereof or amino acids which are not generallyrecognized to be part of any specific protein sequence. Examples of suchfusion proteins are immunogenic polypeptides, proteins with longcirculating half lives, such as immunoglobulin constant regions, markerproteins, proteins or polypeptides that facilitate purification of thedesired peptide or peptibody, and polypeptide sequences that promoteformation of multimeric proteins (such as leucine zipper motifs that areuseful in dimer formation/stability).

This type of insertional variant generally has all-or a substantialportion of the native molecule, linked at the N- or C-terminus, to allor a portion of a second polypeptide. For example, fusion proteinstypically employ leader sequences from other species to permit therecombinant expression of a protein in a heterologous host. Anotheruseful fusion protein includes the addition of an immunologically activedomain, such as an antibody epitope, to facilitate purification of thefusion protein. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions.

There are various commercially available fusion protein expressionsystems that may be used in the present invention. Particularly usefulsystems include but are not limited to the glutathione-S-transferase(GST) system (Pharmacia), the maltose binding protein system (NEB,Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6xHissystem (Qiagen, Chatsworth, Calif.). These systems are capable ofproducing recombinant peptides and/or peptibodies bearing only a smallnumber of additional amino acids, which are unlikely to significantlyaffect the activity of the peptide or peptibody. For example, both theFLAG system and the 6xHis system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of a polypeptide to its native conformation. Another N-terminalfusion that is contemplated to be useful is the fusion of a Met-Lysdipeptide at the N-terminal region of the protein or peptides. Such afusion may produce beneficial increases in protein expression oractivity.

Other fusion systems produce polypeptide hybrids where it is desirableto excise the fusion partner from the desired peptide or peptibody. Inone embodiment, the fusion partner is linked to the recombinantpeptibody by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by. the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

The invention also provides fusion polypeptides which comprises all orpart of a peptibody or peptide of the present invention, in combinationwith truncated tissue factor (tTF). tTF is a vascular targeting agentconsisting of a truncated form of a human coagulation-inducing proteinthat acts as a tumor blood vessel clotting agent, as described U.S. Pat.Nos.: 5,877,289; 6,004,555; 6,132,729; 6,132,730; 6,156,321; andEuropean Pat. No. EP 0988056. The fusion of tTF to the anti-Ang-2peptibody or peptide, or fragments thereof facilitates the delivery ofanti-Ang-2 to target cells.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a peptide or peptibody are removed.Deletions can be effected at one or both termini of the peptibody, orfrom removal of one or more residues within the peptibody amino acidsequence. Deletion variants necessarily include all fragments of apeptide or peptibody.

In still another aspect, the invention provides substitution variants ofpeptides and peptibodies of the invention. Substitution variants includethose peptides and peptibodies wherein one or more amino acid residuesare removed and replaced with one or more alternative amino acids, whichamino acids may be naturally occurring or non-naturally occurring.Substitutional variants generate peptides or peptibodies that are“similar” to the original peptide or peptibody, in that the twomolecules have a certain percentage of amino acids that are identical.Substitution variants include substitutions of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, amino acids within a peptide or peptibody,wherein the number of substitutions may be up to ten percent or more, ofthe amino acids of the peptide or peptibody. In one aspect, thesubstitutions are conservative in nature, however, the inventionembraces substitutions that are also non-conservative and also includesunconventional amino acids.

Identity and similarity of related peptides and peptibodies can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press (1987);Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York (1991); and Carillo et al., SIAM J. AppliedMath., 48:1073 (1988).

Preferred methods to determine the relatedness or percent identity oftwo peptides or polypeptides, or a polypeptide and a peptide, aredesigned to give the largest match between the sequences tested. Methodsto determine identity are described in publicly available computerprograms. Preferred computer program methods to determine identitybetween two sequences include, but are not limited to, the GCG programpackage, including GAP (Devereux et al., Nucl. Acid. Res., 12:387(1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.,BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410(1990)). The BLASTX program is publicly available from the NationalCenter for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul etal., supra (1990)). The well-known Smith Waterman algorithm may also beused to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least tenpercent of the full length of the target polypeptide being compared,i.e., at least 40 contiguous amino acids where sequences of at least 400amino acids are being compared, 30 contiguous amino acids wheresequences of at least 300 to about 400 amino acids are being compared,at least 20 contiguous amino acids where sequences of 200 to about 300amino acids are being compared, and at least 10 contiguous amino acidswhere sequences of about 100 to 200 amino acids are being compared.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is typically calculated as 3× the average diagonal; the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., Atlas of ProteinSequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix;Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970);

Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992);

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program may be useful with the above parameters. In certainembodiments, the aforementioned parameters are the default parametersfor polypeptide comparisons (along with no penalty for end gaps) usingthe GAP algorithm.

In certain embodiments, the parameters for polynucleotide moleculesequence (as opposed to an amino acid sequence) comparisons include thefollowing:

Algorithm: Needleman et al., supra (1970);

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program may also be useful with the above parameters. Theaforementioned parameters are the default parameters for polynucleotidemolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be madewill be apparent to those of skill in the art and will depend on thespecific comparison to be made, such as DNA-to-DNA, protein-to-protein,protein-to-DNA; and additionally, whether the comparison is betweengiven pairs of sequences (in which case GAP or BestFit are generallypreferred) or between one sequence and a large database of sequences (inwhich case FASTA or BLASTA are preferred).

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference forany purpose.

The amino acids may have either L or D stereochemistry (except for Gly,which is neither L nor D) and the polypeptides and compositions of thepresent invention may comprise a combination of stereochemistries.However, the L stereochemistry is preferred. The invention also providesreverse molecules wherein the amino terminal to carboxy terminalsequence of the amino acids is reversed. For example, the reverse of amolecule having the normal sequence X₁-X₂-X₃ would be X₃-X₂-X₁. Theinvention also provides retro-reverse molecules wherein, as above, theamino terminal to carboxy terminal sequence of amino acids is reversedand residues that are normally “L” enantiomers are altered to the “D”stereoisomer form.

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include, withoutlimitation: aminoadipic acid, beta-alanine, beta-aminopropionic acid,aminobutyric acid, piperidinic acid, aminocaprioic acid, aminoheptanoicacid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid,desmosine, diaminopimelic acid, diaminopropionic acid, N-ethylglycine,N-ethylaspargine, hyroxylysine, allo-hydroxylysine, hydroxyproline,isodesmosine, allo-isoleucine, N-methylglycine, sarcosine,N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine,4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and amino acids (e.g., 4-hydroxyproline).

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

It will be appreciated that amino acid residues can be divided intoclasses based on their common side chain properties:

-   -   1. Neutral Hydrophobic: Alanine (Ala; A), Valine (Val; V),        Leucine (Leu; L), Isoleucine (Ile; I), Proline (Pro; P),        Tryptophan (Trp; W), Phenylalanine (Phe; F), and Methionine        (Met, M).    -   2. Neutral Polar: Glycine (Gly; G); Serine (Ser; S), Threonine        (Thr; T), Tyrosine (Tyr; Y), Cysteine (Cys; C), Glutamine (Glu;        Q), Asparagine (Asn; N), and Norleucine.    -   3. Acidic: Aspartic Acid (Asp; D), Glutamic Acid (Glu; E);    -   4) Basic: Lysine (Lys; K), Arginine (Arg; R), Histidine (His;        H).        See Lewin, B., Genes V, Oxford University Press (1994), p. 11.

Conservative amino acid substitutions may encompass unconventional aminoacid residues, which are typically incorporated by chemical peptidesynthesis rather than by synthesis in biological systems. These include,without limitation, peptidomimetics and other reversed or inverted formsof amino acid moieties. Non-conservative substitutions may involve theexchange of a member of one of these classes for a member from anotherclass.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional peptibody or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 2 below.

TABLE 2 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln, Glu, Asp Gln Asp Glu, Gln, Asp Glu Cys Ser, Ala Ser Gln Asn,Glu, Asp Asn Glu Asp, Gln, Asn Asp Gly Pro, Ala Ala His Asn, Gln, Lys,Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu Norleucine Leu Norleucine, Ile,Val, Met, Ile Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, AsnMet Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly SerThr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, SerPhe Val Ile, Met, Leu, Phe, Ala, Leu Norleucine

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In certainembodiments, one can identify residues and portions of the moleculesthat are conserved among similar peptides or polypeptides. In certainembodiments, even areas that may be important for biological activity orfor structure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art may opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. In certain embodiments, one skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change may be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, peptibody variants include glycosylationvariants wherein one or more glycosylation sites, such as a N-linkedglycosylation site, has been added to the peptibody. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution or addition of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions which eliminate this sequence will remove an existingN-linked carbohydrate chain. Also provided is a rearrangement ofN-linked carbohydrate chains wherein one or more N-linked glycosylationsites (typically those that are naturally occurring) are eliminated andone or more new N-linked sites are created.

The invention also provides “derivatives” that include peptibodiesbearing modifications other than, or in addition to, insertions,deletions, or substitutions of amino acid residues. Preferably, themodifications are covalent in nature, and include for example, chemicalbonding with polymers, lipids, other organic, and inorganic moieties.Derivatives of the invention may be prepared to increase circulatinghalf-life of a peptibody, or may be designed to improve targetingcapacity for the peptibody to desired cells, tissues, or organs.

Exemplary derivatives include moieties wherein one or more of thefollowing modifications have been made:

-   -   One or more of the peptidyl [—C(O)NR—] linkages (bonds) have        been replaced by a non-peptidyl linkage such as a —CH₂-carbamate        linkage [—CH₂—OC(O)NR—]; a phosphonate linkage; a        —CH₂-sulfonamide [—CH₂—S(O)₂NR—] linkage; a urea [—NHC(O)NH—]        linkage; a —CH₂-secondary amine linkage; or an alkylated        peptidyl linkage [—C(O)NR⁶— where R⁶ is lower alkyl];    -   Peptides wherein the N-terminus is derivatized to a —NRR¹ group;        to a —NRC(O)R group; to a —NRC(O)OR group; to a —NRS(O)₂R group;        to a —NHC(O)NHR group, where R and R¹ are hydrogen or lower        alkyl, with the proviso that R and R¹ are not both hydrogen; to        a succinimide group; to a benzyloxycarbonyl-NH— (CBZ—NH—) group;        or to a benzyloxycarbonyl-NH— group having from 1 to 3        substituents on the phenyl ring selected from the group        consisting of lower alkyl, lower alkoxy, chloro, and bromo; and    -   Peptides wherein the free C terminus is derivatized to —C(O)R²        where R² is selected from the group consisting of lower alkoxy        and —NR³R⁴ where R³ and R⁴ are independently selected from the        group consisting of hydrogen and lower alkyl. By “lower” is        meant a group having from 1 to 6 carbon atoms.

Additionally, modifications of individual amino acids may be introducedinto the polypeptides or compositions of the invention by reactingtargeted amino acid residues of the peptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. The following are exemplary:

Lysinyl and amino terminal residues may be reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineguanidino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane may be used to formi O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutarnyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular carriers. Commonly used cross-linkingagents include, e.g., 1,1 -bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 may be employed for protein immobilization.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains(Creighton, T. E., Proteins: Structure and Molecule Properties, W. H.Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN-terminal amine, and, in some instances, amidation of the C-terminalcarboxyl groups.

Such derivatized moieties preferably improve one or more characteristicsincluding anti-angiogenic activity, solubility, absorption, biologicalhalf life, and the like of the compounds. Alternatively, derivatizedmoieties may result in compounds that have the same, or essentially thesame, characteristics and/or properties of the compound that is notderivatized. The moieties may alternatively eliminate or attenuate anyundesirable side effect of the compounds and the like.

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes. Thus, allmodifications, substitution, derivitizations, etc. discussed hereinapply equally to all aspects of the present invention, including but notlimited to peptides, peptide dimers and multimers, linkers, andvehicles.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar peptides that are important foractivity or structure. In view of such a comparison, one can predict theimportance of amino acid residues in a peptide that correspond to aminoacid residues that are important for activity or structure in similarpeptides. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues ofthe peptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of a peptide with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution ateach desired amino acid residue. The variants can then bescreened using activity assays know to those skilled in the art. Suchdata could be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974); Chouet al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv. Enzymol.Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev.Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979).Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl etal., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al.,Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-8(1987)), and “evolutionary linkage” (See Home, supra, and Brenner,supra).

The invention further embraces derivative specific binding agents, e.g.peptibodies, covalently modified to include one or more water solublepolymer attachments, such as polyethylene glycol, polyoxyethyleneglycol, or polypropylene glycol, as described U.S. Pat. Nos.: 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. Still otheruseful polymers known in the art include monomethoxy-polyethyleneglycol, dextran, cellulose, or other carbohydrate based polymers,poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycolhomopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as wellas mixtures of these polymers. Particularly preferred are peptibodiescovalently modified with polyethylene glycol (PEG) subunits.Water-soluble polymers may be bonded at specific positions, for exampleat the amino terminus of the peptibodies, or randomly attached to one ormore side chains of the polypeptide. The use of PEG for improving thetherapeutic capacity for specific binding agents, e.g. peptibodies, andfor humanized antibodies in particular, is described in U.S. Pat. No.6,133,426 to Gonzales et al., issued Oct. 17, 2000.

The invention also contemplates derivatizing the peptide and/or vehicleportion of the compounds. Such derivatives may improve the solubility,absorption, biological half-life, and the like of the compounds. Themoieties may alternatively eliminate or attenuate any undesirableside-effect of the compounds and the like. Exemplary derivatives includecompounds in which:

1. The compound or some portion thereof is cyclic. For example, thepeptide portion may be modified to contain two or more Cys residues(e.g., in the linker), which could cyclize by disulfide bond formation.

2. The compound is cross-linked or js rendered capable of cross-linkingbetween molecules. For example, the peptide portion may be modified tocontain one Cys residue and thereby be able to form an intermoleculardisulfide bond with a like molecule. The compound may also becross-linked through its C-terminus.

3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by anon-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂-carbamate[—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide-[—CH₂—S(O)₂NR—], urea[—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR₆—wherein R₆ is lower alkyl].

4. The N-terminus is derivatized. Typically, the N-terminus may beacylated or modified to a substituted amine. Exemplary N-terminalderivative groups include —NRR₁ (other than —NH₂), —NRC(O)R₁,—NRC(O)OR₁, —NRS(O)₂R₁, —NHC(O)NHR₁, succinimide, orbenzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R1 are each independentlyhydrogen or lower alkyl and wherein the phenyl ring may be substitutedwith 1 to 3 substituents selected from the group consisting of C₁-C₄alkyl, C₁-C₄ alkoxy, chloro, and bromo.

5. The free C-terminus is derivatized. Typically, the C-terminus isesterified or amidated. For example, one may use methods described inthe art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention at theC-terminus. Likewise, one may use methods described in the art to add—NH₂ to compounds of this invention at the C-terminus. ExemplaryC-terminal derivative groups include, for example, —C(O)R₂ wherein R₂ islower alkoxy or —NR₃R₄ wherein R₃ and R₄ are independently hydrogen orC₁-C₈ alkyl (preferably C₁-C₄ alkyl).

6. A disulfide bond is replaced with another, preferably more stable,cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar (supra);Alberts et al., Thirteenth Am. Pep. Symp., 357-9 (1993).

7. One or more individual amino acid residues is modified. Variousderivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl side chain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar, (supra).

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains[Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983)].

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Affinity Maturation

One embodiment of the present invention includes “affinity matured”peptides and peptibodies. This procedure contemplates increasing theaffinity or the bio-activity of the peptides and peptibodies of thepresent invention using phage display or other selection technologies.Based on a consensus sequence (which is generated for a collection ofrelated peptides), directed secondary phage display libraries can begenerated in which the “core” amino acids (determined from the consensussequence) are held constant or are biased in frequency of occurrence.Alternatively, an individual peptide sequence can be used to generate abiased, directed phage display library. Panning of such libraries canyield peptides (which can be converted to peptibodies) with enhancedbinding to Ang-2 or with enhanced bio-activity.

Non-Peptide Analogs/Protein Mimetics

Furthermore, non-peptide analogs of peptides that provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be. prepared based on a selected inhibitory peptideby replacement of one or more residues by nonpeptide moieties.Preferably, the nonpeptide moieties permit the peptide to retain itsnatural confirmation, or stabilize a preferred, e.g., bioactive,confirmation which retains the ability to recognize and bind Ang-2. Inone aspect, the resulting analog/mimetic exhibits increased bindingaffinity for Ang-2. One example of methods for preparation of nonpeptidemimetic analogs from peptides is described in Nachman et al., Regul.Pept. 57:359-370 (1995). If desired, the peptides of the invention canbe modified, for instance, by glycosylation, amidation, carboxylation;or phosphorylation, or by the creation of acid addition salts, amides,esters, in particular C-terminal esters, and N-acyl derivatives of thepeptides of the invention. The peptibodies also can be modified tocreate peptide derivatives by forming covalent or noncovalent complexeswith other moieties. Covalently-bound complexes can be prepared bylinking the chemical moieties to functional groups on the side chains ofamino acids comprising the peptibodies, or at the N- or C-terminus.

In particular, it is anticipated that the peptides can be conjugated toa reporter group, including, but not limited to a radiolabel, afluorescent label, an enzyme (e.g., that catalyzes a colorimetric orfluorometric reaction), a substrate, a solid matrix, or a carrier (e.g.,biotin or avidin). The invention accordingly provides a moleculecomprising a peptibody molecule, wherein the molecule preferably furthercomprises a reporter group selected from the group consisting of aradiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix,and a carrier. Such labels are well known to those of skill in the art,e.g., biotin labels are particularly contemplated. The use of suchlabels is well known to those of skill in the art and is described in,e.g., U.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; and 4,277,437.Other labels that will be useful include but are not limited toradioactive labels, fluorescent labels and chemiluminescent labels. U.S.Patents concerning use of such labels include, for example, U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; and 3,996,345. Any of thepeptibodies of the present invention may comprise one, two, or more ofany of these labels.

Methods of Making Peptides

The peptides of the present invention can be generated using a widevariety of techniques known in the art. For example, such peptides canbe synthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young (supra); Tam et al., J. Am. Chem. Soc.,105:6442, (1983); Merrifield, Science 232:341-347 (1986); Barany andMerrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, NewYork, 1-284; Barany et al., Int. J. Pep. Protein Res., 30:705-739(1987); and U.S. Pat. No. 5,424,398, each incorporated herein byreference.

Solid phase peptide synthesis methods use acopoly(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer.These methods for peptide synthesis use butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxy-carbonyl(FMOC) protection of alpha-amino groups.Both methods involve stepwise syntheses whereby a single amino acid isadded at each step starting from the C-terminus of the peptide (See,Coligan et al., Curr. Prot. Immunol., Wiley Interscience, 1991, Unit 9).On completion of chemical synthesis, the synthetic peptide can bedeprotected to remove the t-BOC or FMOC amino acid blocking groups andcleaved from the polymer by treatment with acid at reduced temperature(e.g., liquid HF-10% anisole for about 0.25 to about 1 hours at 0° C.).After evaporation of the reagents, the peptides are extracted from thepolymer with 1% acetic acid solution that is then lyophilized to yieldthe crude material. This can normally be purified by such techniques asgel filtration on Sephadex G-15 using 5% acetic acid as a solvent.Lyophilization of appropriate fractions of the column will yield thehomogeneous peptides or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by the solid phase Edman degradation.

Other methods, such as selecting peptides from a phage display library,are also available. Libraries can be prepared from sets of amino acidsas described herein. Phage display can be particularly effective inidentifying peptides useful according to the invention. Briefly, oneprepares a phage library (using e.g. ml 13, fd, or lambda phage),displaying inserts from 4 to about 80 amino acid residues. The insertsmay represent, for example, a completely degenerate or biased array. Onethen can select phage-bearing inserts that bind to the desired antigen.This process can be repeated through several cycles of reselection ofphage that bind to the desired antigen. Repeated rounds lead toenrichment of phage bearing particular sequences. DNA sequence analysiscan be conducted to identify the sequences of the expressed peptides.The minimal linear portion of the sequence that binds to the desiredantigen can be determined. One can repeat the procedure using a biasedlibrary containing inserts containing part or all of the minimal linearportion plus one or more additional degenerate residues upstream ordownstream thereof. These techniques may identify peptides of theinvention with still greater binding affinity for Ang-2 than agentsalready identified herein.

Regardless of the manner in which the peptides are prepared, a nucleicacid molecule encoding each such peptide and peptibody can be generatedusing standard recombinant DNA procedures. The nucleotide sequence ofsuch DNA molecules can be manipulated as appropriate without changingthe amino acid sequence they encode to account for the degeneracy of thenucleic acid code as well as to account for codon preference inparticular host cells.

Recombinant DNA techniques are a convenient method for preparing fulllength peptibodies and other large proteinaceous specific binding agentsof the present invention, or fragments thereof. A DNA molecule encodingthe peptibody or fragment may be inserted into an expression vector,which can in turn be inserted into a host cell for production of theantibody or fragment.

Generally, a DNA molecule encoding a peptide or peptibody can beobtained using procedures described herein in the Examples. Probes andtypical hybridization conditions are those such as set forth in Ausubelet al. (Current Protocols in Molecular Biology, Current Protocols Press[1994]). After hybridization, the probed blot can be washed at asuitable stringency, depending on such factors as probe size, expectedhomology of probe to clone, the type of library being screened, and thenumber of clones being screened. Examples of high stringency screeningare 0.1×SSC, and 0.1 percent SDS at a temperature between 50-65° C.

Yeast two-hybrid screening methods also may be used to identify peptidesof the invention that bind to the Ang-2. Thus, antigen, or a fragmentthereof, can be used to screen peptide libraries, including phagedisplay libraries, to identify and select Ang-2 binding agents, e.g.peptibodies, of the present invention.

Alternatively, a variety of expression vector/host systems may beutilized to contain and express the peptides of the invention. Thesesystems include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells that are usefulin recombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of thepeptides are described herein below.

The term “expression vector” refers to a plasmid, phage, virus orvector, for expressing a polypeptide from a DNA (RNA) sequence. Anexpression vector can comprise a transcriptional unit comprising anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, promoters or enhancers, (2) astructural or sequence that encodes the binding agent which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant protein isexpressed without a leader or transport sequence, it may include anamino terminal methionyl residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal peptide product.

For example, the peptides may be recombinantly expressed in yeast usinga commercially available expression system, e.g., the Pichia ExpressionSystem (Invitrogen, San Diego, Calif.), following the manufacturer'sinstructions. This system also relies on the pre-pro-alpha sequence todirect secretion, but transcription of the insert is driven by thealcohol oxidase (AOX1) promoter upon induction by methanol.

The secreted peptide is purified from the yeast growth medium by, e.g.,the methods used to purify the peptide from bacterial and mammalian cellsupernatants.

Alternatively, the cDNA encoding the peptide may be cloned into thebaculovirus expression vector pVL1393 (PharMingen, San Diego, Calif.).This vector can be used according to the manufacturer's directions(PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-freemedia and to produce recombinant protein. The recombinant protein can bepurified and concentrated from the media using a heparin-Sepharosecolumn (Pharmacia).

Alternatively, the peptide may be expressed in an insect system. Insectsystems for protein expression are well known to those of skill in theart. In one such system, Autographa califomica nuclear polyhedrosisvirus (AcNPV) can be used as a vector to express foreign genes inSpodoptera frugiperda cells or in Trichoplusia larvae. The peptidecoding sequence can be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the peptide will render the polyhedringene inactive and produce recombinant virus lacking coat protein coat.The recombinant viruses can be used to infect S. frugiperda cells orTrichoplusia larvae in which the peptide is expressed. Smith et al., J.Virol. 46: 584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA) 91:3224-7 (1994).

In another example, the DNA sequence encoding the peptide can beamplified by PCR and cloned into an appropriate vector for example,pGEX-3X (Pharmacia). The pGEX vector is designed to produce a fusionprotein comprising glutathione-S-transferase (GST), encoded by thevector, and a protein encoded by a DNA fragment inserted into thevector's cloning site. The primers for PCR can be generated to includefor example, an appropriate cleavage site. Where the fusion moiety isused solely to facilitate expression or is otherwise not desirable as anattachment to the peptide of interest, the recombinant fusion proteinmay then be cleaved from the GST portion of the fusion protein. ThepGEX-3X/specific binding agent peptide construct is transformed into E.coli XL-1 Blue cells (Stratagene, La Jolla Calif.), and individualtransformants isolated and grown. Plasmid DNA from individualtransformants can be purified and partially sequenced using an automatedsequencer to confirm the presence of the desired specific binding agentencoding nucleic acid insert in the proper orientation.

Certain peptide compositions of the present invention are those in whicha peptibody is conjugated to any anti-tumor peptide such as tumornecrosis factor (TNF). In a particularly preferred method, theTNF-specific binding agent peptides chimeras are generated asrecombinant fusions with peptide-encoding sequences fused in frame toTNF (Novagen, Madison, Wis.) encoding sequences. Peptide-TNF cDNA can becloned into pET-11b vector (Novagen) and the expression of TNF-peptidesin BL21 E. coli can be induced according to the pETI lb manufacturer'sinstruction. Soluble TNF-peptides can be purified from bacterial lysatesby ammonium sulfate preparation, hydrophobic interaction chromatographyon Phenyl-Sepharose 6 Fast Flow, ion exchange chromatography onDEAE-Sepharose Fast Flow and gel filtration chromatography onSephacryl-S-300 HR.

The fusion protein, which may be produced as an insoluble inclusion bodyin the bacteria, can be purified as follows. Host cells can besacrificed by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St. Louis, Mo.) for15 minutes at room temperature. The lysate can be cleared by sonication,and cell debris can be pelleted by centrifugation for 10 minutes at12,000×g. The fusion protein-containing pellet can be resuspended in 50mM Tris, pH 8, and 10 mM EDTA,. layered over 50% glycerol, andcentrifuged for 30 minutes at 6000×g. The pellet can be resuspended instandard phosphate buffered saline solution (PBS) free of Mg++ and Ca++.The fusion protein can be further purified by fractionating theresuspended pellet in a denaturing SDS-PAGE (Sambrook et al., supra).The gel can be soaked in 0.4 M KCl to visualize the protein, which canbe excised and electroeluted in gel-running buffer lacking SDS. If theGST/fusion protein is produced in bacteria as a soluble protein, it canbe purified using the GST Purification Module (Pharmacia).

The fusion protein may be subjected to digestion to cleave the GST fromthe peptide of the invention. The digestion reaction (2040 mg fusionprotein, 20-30 units human thrombin (4000 U/mg, Sigma) in 0.5 ml PBS canbe incubated 16-48 hrs at room temperature and loaded on a denaturingSDS-PAGE gel to fractionate the reaction products. The gel can be soakedin 0.4 M KCl to visualize the protein bands. The identity of the proteinband corresponding to the expected molecular weight of the peptide canbe confirmed by amino acid sequence analysis using an automatedsequencer (Applied Biosystems Model 473A, Foster City, Calif.).Alternatively, the identity can be confirmed by performing HPLC and/ormass spectometry of the peptides.

Alternatively, a DNA sequence encoding the peptide can be cloned into aplasmid containing a desired promoter and, optionally, a leader sequence[Better et al., Science 240:1041-43 (1988)]. The sequence of thisconstruct can be confirmed by automated sequencing. The plasmid can thenbe transformed into E. coli strain MC 1061 using standard proceduresemploying CaCl2 incubation and heat shock treatment of the bacteria(Sambrook et al., supra). The transformed bacteria can be grown in LBmedium supplemented with carbenicillin, and production of the expressedprotein can be induced by growth in a suitable medium. If present, theleader sequence can effect secretion of the peptide and be cleavedduring secretion.

The secreted recombinant protein can be purified from the bacterialculture media by the methods described herein below.

Mammalian host systems for the expression of the recombinant protein arewell known to those of skill in the art. Host cell strains can be chosenfor a particular ability to process the expressed protein or producecertain post-translation modifications that will be useful in providingprotein activity. Such modifications of the protein include, but are notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation. Different host cells such as CHO, HeLa, MDCK,293, W138, and the like have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of theintroduced, foreign protein.

It is preferable that the transformed cells be used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellscan be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems can be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thyrnidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for DHFR which confers resistance to methotrexate; gptwhich confers resistance to mycophenolic acid; neo which confersresistance to the aminoglycoside G418 and confers resistance tochlorsulfuron; and hygro which confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identificatiori of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Purification and Refolding of Specific Binding Agents

In some cases, the specific binding agents such as the peptides and/orpeptibodies of this invention may need to be “refolded” and oxidizedinto a proper tertiary structure and generating disulfide linkages inorder to be biologically active. Refolding can be accomplished using anumber of procedures well known in the art. Such methods include, forexample, exposing the solubilized polypeptide agent to a pH usuallyabove 7 in the presence of a chaotropic agent. The selection ofchaotrope is similar to the choices used for inclusion bodysolubilization, however a chaotrope is typically used at a lowerconcentration. An exemplary chaotropic agent is guanidine. In mostcases, the refolding/oxidation solution will also contain a reducingagent plus its oxidized form in a specific ratio to generate aparticular redox potential which allows for disulfide shuffling to occurfor the formation of cysteine bridges. Some commonly used redox couplesinclude cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride,dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME.In many instances, a co-solvent may be used to increase the efficiencyof the refolding. Commonly used cosolvents include glycerol,polyethylene gluycol of various molecular weights, and arginine.

It may be desirable to purify the peptides and peptibodies of thepresent invention. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the proteinaceous and non-proteinaceousfractions. Having separated the peptide and/or peptibody from otherproteins, the peptide or polypeptide of interest can be further purifiedusing chromatographic and electrophoretic techniques to achieve partialor complete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of peptibodies andpeptides or the present invention are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of a peptibodyor peptide of the present invention. The term “purified peptibody orpeptide” as used herein, is intended to refer to a composition,isolatable from other components, wherein the peptibody or peptide ispurified to any degree relative to its naturally-obtainable state. Apurified peptide or peptibody therefore also refers to a peptibody orpeptide that is free from the environment in which it may naturallyoccur.

Generally, “purified” will refer to a peptide or peptibody compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a peptide or peptibody composition inwhich the peptibody or peptide forms the major component of thecomposition, such as constituting about 50%, about 60%, about 70%, about80%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of thepeptide or peptibody will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific binding activity of an active fraction, or assessing the amountof peptide or peptibody within a fraction by SDS/PAGE analysis. Apreferred method for assessing the purity of a peptide or peptibodyfraction is to calculate the binding activity of the fraction, tocompare it to the binding activity of the initial extract, and to thuscalculate the degree of purification, herein assessed by a “-foldpurification number.” The actual units used to represent the amount ofbinding activity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not thepeptibody or peptide exhibits a detectable binding activity.

Various techniques suitable for use in purification will be well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies (immunoprecipitation) and thelike or by heat denaturation, followed by centrifugation; chromatographysteps such as affinity chromatography (e.g., Protein-A-Sepharose), ionexchange, gel filtration, reverse phase, hydroxylapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed. or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified specific binding agent.

There is no general requirement that the peptide or peptibody of thepresent invention always be provided in its most purified state. Indeed,it is contemplated that less substantially specific binding agentproducts will have utility in certain embodiments. Partial purificationmay be accomplished by using fewer purification steps in combination, orby utilizing different forms of the same general purification scheme.For example, it is appreciated that a cation-exchange columnchromatography performed utilizing an HPLC apparatus will generallyresult in a greater “-fold” purification than the same techniqueutilizing a low-pressure chromatography system. Methods exhibiting alower degree of relative purification may have advantages in totalrecovery of the peptide or peptibody, or in maintaining binding activityof the peptide or peptibody.

It is known that the migration of a peptide or polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE [Capaldiet al., Biochem. Biophys. Res. Comm., 76: 425 (1977)]. It will thereforebe appreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified specificbinding agent expression products may vary.

Binding Assays

Immunological binding assays typically utilize a capture agent to bindspecifically to and often immobilize the analyte target antigen. Thecapture agent is a moiety that specifically binds to the analyte. In oneembodiment of the present invention, the capture agent is a peptide orpeptibody or fragment thereof that specifically binds Ang-2. Theseimmunological binding assays are well known in the art [Asai, ed.,Methods in Cell Biology, Vol. 37, Antibodies in Cell Biology, AcademicPress, Inc., New York (1993)].

Immunological binding assays frequently utilize a labeling agent thatwill signal the existence of the bound complex formed by the captureagent and antigen. The labeling agent can be one of the moleculescomprising the bound complex; i.e. it can be a labeled specific bindingagent or a labeled anti-specific binding agent antibody. Alternatively,the labeling agent can be a third molecule, commonly another antibody,which binds to the bound complex. The labeling agent can be, forexample, an anti-specific binding agent antibody bearing a label. Thesecond antibody, specific for the bound complex, may lack a label, butcan be bound by a fourth molecule specific to the species of antibodieswhich the second antibody is a member of. For example, the secondantibody can be modified with a detectable moiety, such as biotin, whichcan then be bound by a fourth molecule, such as enzyme-labeledstreptavidin. Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the labeling agent. These binding proteins are normalconstituents of the cell walls of streptococcal bacteria and exhibit astrong non-immunogenic reactivity with immunoglobulin constant regionsfrom a variety of species. Akerstrom, J. Immunol., 135:2589-2542 (1985);Chaubert, Mod. Pathol., 10:585-591 (1997).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures.

A. Non-competitive Binding Assays:

Immunological binding assays can be of the non-competitive type. Theseassays have an amount of captured analyte that is directly measured. Forexample, in one preferred “sandwich” assay, the capture agent (antibodyor peptibody) can be bound directly to a solid substrate where it isimmobilized. These immobilized capture agents then capture (bind to)antigen present in the test sample. The protein thus immobilized is thenbound to a labeling agent, such as a second antibody having a label. Inanother preferred “sandwich” assay, the second antibody lacks a label,but can be bound by a labeled antibody specific for antibodies of thespecies from which the second antibody is derived. The second antibodyalso can be modified with a detectable moiety, such as biotin, to whicha third labeled molecule can specifically bind, such as streptavidin.See Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14, Cold SpringHarbor Laboratory, N.Y. (1988), incorporated herein by reference.

B. Competitive Binding Assays:

Immunological binding assays can be of the competitive type. The amountof analyte present in the sample is measure indirectly by measuring theamount of an added analyte displaced, or competed away, from a captureagent (antibody or peptibody) by the analyte present in the sample. Inone preferred competitive binding assay, a known amount of analyte,usually labeled, is added to the sample and the sample is then contactedwith the capture agent. The amount of labeled analyze bound to theantibody is inversely proportional to the concentration of anal ytepresent in the sample (See, Harlow and Lane, Antibodies, A LaboratoryManual, Ch 14, pp. 579-583, supra).

In another preferred competitive binding assay, the capture agent isimmobilized on a solid substrate. The amount of protein bound to thecapture agent may be determined either by measuring the amount ofprotein present in a protein/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount ofprotein may be detected by providing a labeled protein. Harlow and Lane(supra).

Yet another preferred competitive binding assay, hapten inhibition isutilized. Here, a known analyte is immobilized on a solid substrate. Aknown. amount of antibody is added to the sample, and the sample iscontacted with the immobilized analyte. The amount of antibody bound tothe immobilized analyte is inversely proportional to the amount ofanalyte present in the sample. The amount of immobilized antibody may bedetected by detecting either the immobilized fraction of antibody or thefraction that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

C. Utilization of Competitive Binding Assays:

The competitive binding assays can be used for cross-reactivitydeterminations to permit a skilled artisan to determine if a protein orenzyme complex which is recognized by a peptibody of the invention isthe desired protein and not a cross-reacting molecule or to determinewhether the peptibody is specific for the antigen and does not bindunrelated antigens. In assays of this type, antigen can be immobilizedto a solid support and an unknown protein mixture is added to the assay,which will compete with the binding of the peptibodies to theimmobilized protein. The competing molecule also binds one or moreantigens unrelated to the antigen. The ability of the proteins tocompete with the binding of the peptibodies to the immobilized antigenis compared to the binding by the same protein that was immobilized tothe solid support to determine the cross-reactivity of the protein mix.

D. Other Binding Assays

The present invention also provides Western blot methods to detect orquantify the presence of Ang-2 in a sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight and transferring the proteins to a suitable solidsupport, such as nitrocellulose filter, a nylon filter, or derivatizednylon filter. The sample is incubated with peptibodies or fragmentsthereof that specifically bind Ang-2 and the resulting complex isdetected. These peptibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies that specifically bindto the peptibody.

Diagnostic Assays

The derivative binding agents, such as peptides and peptibodies orfragments thereof, of the present invention are useful for the diagnosisof conditions or diseases characterized by expression of Ang-2 orsubunits, or in assays to monitor patients being treated with inducersof Ang-2, its fragments, agonists or inhibitors of Ang-2 activity.Diagnostic assays for Ang-2 include methods utilizing a peptibody and alabel to detect Ang-2 in human body fluids or extracts of cells ortissues. The peptibodies of the present invention can be used with orwithout modification. In a preferred diagnostic assay, the peptibodieswill be labeled by attaching, e.g., a label or a reporter molecule. Awide variety of labels and reporter molecules are known, some of whichhave been already described herein. In particular, the present inventionis useful for diagnosis of human disease.

A variety of protocols for measuring Ang-2 proteins using peptibodiesspecific for the respective protein are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA) and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on Ang-2 is preferred, but a competitivebinding assay can be employed. These assays are described, for example,in Maddox et al., J. Exp. Med., 158:1211 (1983).

In order to provide a basis for diagnosis, normal or standard values forhuman Ang-2 expression are usually established. This determination canbe accomplished by combining body fluids or cell extracts from normalsubjects, preferably human, with a peptibody to Ang-2, under conditionssuitable for complex formation that are well known in the art. Theamount of standard complex formation can be quantified by comparing thebinding of the peptibodies to known quantities of Ang-2 protein, withboth control and disease samples. Then, standard values obtained fromnormal samples can be compared with values obtained from samples fromsubjects potentially affected by disease. Deviation between standard andsubject values suggests a role for Ang-2 in the disease state.

For diagnostic applications, in certain embodiments peptibodies orpeptides of the present invention typically will be labeled with adetectable moiety. The detectable moiety can be any one that is capableof producing, either directly or indirectly, a detectable signal. Forexample, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, suchas alkaline phosphatase, β-galactosidase, or horseradish peroxidase.Bayer et al., Meth. Enz., 184: 138-163, (1990).

Diseases

The present invention provides a binding agent such as a peptide,peptibody, or fragment, variant or derivative thereof that binds toAng-2 that is useful for the treatment of human diseases andpathological conditions. Agents that modulate Ang-2 binding activity, orother cellular activity, may be used in combination with othertherapeutic agents to enhance their therapeutic effects or decreasepotential side effects.

In one aspect, the present invention provides reagents and methodsuseful for treating diseases and conditions characterized by undesirableor aberrant levels of Ang-2 activity in a cell. These diseases includecancers, and other hyperproliferative conditions, such as hyperplasia,psoriasis, contact dermatitis, immunological disorders, and infertility.

The present invention also provides methods of treating cancer in ananimal, including humans, comprising administering to the animal aneffective amount of a specific binding agent, such as a peptibody, thatinhibits or decreases Ang-2 activity. The invention is further directedto methods of inhibiting cancer cell growth, including processes ofcellular proliferation, invasiveness, and metastasis in biologicalsystems. Methods include use of a compound of the invention as aninhibitor of cancer cell growth. Preferably, the methods are employed toinhibit or reduce cancer cell growth, invasiveness, metastasis, or tumorincidence in living animals, such as mammals. Methods of the inventionare also readily adaptable for use in assay systems, e.g., assayingcancer cell growth and properties thereof, as well as identifyingcompounds that affect cancer cell growth.

The cancers treatable by methods of the present invention preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed malignant and may lead todeath of the organism. Malignant neoplasms or cancers are distinguishedfrom benign growths in that, in addition to exhibiting aggressivecellular proliferation, they may invade surrounding tissues andmetastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater dedifferentiation),and of their organization relative to one another and their surroundingtissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include.solid tumors,i.e., carcinomas and sarcomas. Carcinomas include those malignantneoplasms derived from epithelial cells that infiltrate (invade) thesurrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or which form recognizableglandular structures. Another broad category or cancers includessarcomas, which are tumors whose cells are embedded in a fibrillar orhomogeneous substance like embryonic connective tissue. The inventionalso enables treatment of cancers of the myeloid or lymphoid systems,including leukemias, lymphomas and other cancers that typically do notpresent as a tumor mass, but are distributed in the vascular orlymphoreticular systems.

The type of cancer or tumor cells amenable to treatment according to theinvention include, for example, ACTH-producing tumor, acute lymphocyticleukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex,bladder cancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and non-small cell), malignant peritonealeffusion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma,ovarian cancer, ovarian (germ cell) cancer, pancreatic cancer, penilecancer, prostate cancer, retinoblastoma, skin cancer, soft tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, uterine cancer, vaginal cancer,cancer of the vulva, and Wilms' tumor.

The invention is particularly illustrated herein in reference totreatment of certain types of experimentally defined cancers. In theseillustrative treatments, standard state-of-the-art in vitro and in vivomodels have been used. These methods can be used to identify agents thatcan be expected to be efficacious in in vivo treatment regimens.However, it will be understood that the method of the invention is notlimited to the treatment of these tumor types, but extends to any solidtumor derived from any organ system. Cancers whose invasiveness ormetastasis is associated with Ang-2 expression or activity areespecially susceptible to being inhibited or even induced to regress bymeans of the invention.

The invention can also be practiced by including with a compound of theinvention such as a peptibody in combination with another anti-cancerchemotherapeutic agent, such as any conventional chemotherapeutic agent.The combination of a specific binding agent with such other agents canpotentiate the chemotherapeutic protocol. Numerous chemotherapeuticprotocols will present themselves in the mind of the skilledpractitioner as being capable of incorporation into the method of theinvention. Any chemotherapeutic agent can be used, including alkylatingagents, antimetabolites, hormones and antagonists, radioisotopes, aswell as natural products. For example, the compound of the invention canbe administered with antibiotics such as doxorubicin and otheranthracycline analogs, nitrogen mustards such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinoma of thebreast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the compound can be administered inconjunction with leuprolide or goserelin (synthetic peptide analogs ofLH-RH). Other antineoplastic protocols include the use of a tetracyclinecompound with another treatment modality, e.g., surgery, radiation,etc., also referred to herein as “adjunct antineoplastic modalities.”Thus, the method of the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

The present invention thus provides compositions and methods useful forthe treatment of a wide variety of cancers, including solid tumors andleukemias. Types of cancer that may be treated include, but are notlimited to: adenocarcinoma of the breast, prostate, and colon; all formsof bronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma;neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignantcarcinoid syndrome; carcinoid heart disease; carcinoma (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2,merkel cell, mucinous, non-small cell lung, oat cell, papillary,scirrhous, bronchiolar, bronchogenic, squamous cell, and transitionalcell); histiocytic disorders; leukemia; histiocytosis malignant;Hodgkin's disease; immunoproliferative small lung cell carcinoma;non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;ameloblastoma; cementoma; odontoma; teratoma; thymoma; tophoblastictumor. Further, the following types of cancers may also. be treated:adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma;cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma;hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertolicell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma;myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma;ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma;neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma;paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasiawith eosinophilia; angioma sclerosing; angiomatosis; glomangioma;hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma;lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma;carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervicaldysplasia.

Another aspect of the present invention is using the materials andmethods of the present invention to prevent and/or treat anyhyperproliferative condition of the skin including psoriasis and contactdermatitis or other hyperproliferative diseases. It has beendemonstrated that patients with psoriasis and contact dermatitis haveelevated Ang-2 activity within these lesions [Ogoshi et al., J. Inv.Dennatol., 110:818-23 (1998)]. Preferably, specific binding agentsspecific for Ang-2 will be used in combination with other pharmaceuticalagents to treat humans that express these clinical symptoms. Thespecific binding agents can be delivered using any of the variouscarriers through routes of administration described herein and othersthat are well known to those of skill in the art.

Other aspects of the present invention include treating variousretinopathies (including diabetic retinopathy and age-related maculardegeneration) in which angiogenesis is involved, as well asdisorders/diseases of the female reproductive tract such asendometriosis, uterine fibroids, and other such conditions associatedwith dysfunctional vascular proliferation (including endometrialmicrovascular growth) during the female reproductive cycle.

Still another aspect of the present invention relates to treatingabnormal vascular growth including cerebral arteriovenous malformations(AVMs) gastrointestinal mucosal injury and repair, ulceration of thegastroduodenal mucosa in patients with a history of peptic ulcerdisease, including ischemia resulting from stroke, a wide spectrum ofpulmonary vascular disorders in liver disease and portal hypertension inpatients with nonhepatic portal hypertension.

Another aspect of present invention is the prevention of cancersutilizing the compositions and methods provided by the presentinvention. Such reagents will include specific binding agents such aspeptibodies against Ang-2.

Pharmaceutical Compositions

Pharmaceutical compositions of Ang-2 specific binding agents such aspeptibodies are within the scope of the present invention.Pharmaceutical compositions comprising antibodies are described indetail in, for example, U.S. Pat. No. 6,171,586, to Lam et al., issuedJan. 9, 2001. Such compositions comprise a therapeutically orprophylactically effective amount of a specific binding agent, such asan antibody, or a fragment, variant, derivative or fusion thereof asdescribed herein, in admixture with a pharmaceutically acceptable agent.In a preferred embodiment, pharmaceutical compositions compriseantagonist specific binding agents that modulate partially or completelyat least one biological activity of Ang-2 in admixture with apharmaceutically acceptable agent. Typically, the specific bindingagents will be sufficiently purified for administration to an animal.

The pharmaceutical composition may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.Suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates, other organic acids); bulking agents(such as mannitol or glycine), chelating agents [such as ethylenediaminetetraacetic acid (EDTA)]; complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides and other carbohydrates (such as glucose, mannose, ordextrins); proteins (such as serum albumin, gelatin orimrnmunoglobulins); coloring; flavoring and diluting agents; emulsifyingagents; hydrophilic polymers (such as polyvinylpyrrolidone); lowmolecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, PEG, sorbitan esters,polysorbates such as polysorbate 20, polysorbate 80, triton,tromethamine, lecithin, cholesterol, tyloxapal); stability enhancingagents (sucrose or sorbitol); tonicity enhancing agents (such as alkalimetal halides (preferably sodium or potassium chloride, mannitolsorbitol); delivery vehicles; diluents; excipients and/or pharmaceuticaladjuvants. (Remingtori's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the specific binding agent.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefore. In oneembodiment of the present invention, binding agent compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the binding agent product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions may be selected for inhalation or forenteral delivery such as orally, aurally, opthalmically, rectally, orvaginally. The preparation of such pharmaceutically acceptablecompositions is within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired specific binding agent in a pharmaceutically acceptable vehicle.A particularly suitable vehicle for parenteral injection is steriledistilled water in which a binding agent is formulated as a sterile,isotonic solution, properly preserved. Yet another preparation caninvolve the formulation of the desired molecule with an agent, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes, that providesfor the controlled or sustained release of the product which may then bedelivered via a depot injection. Hyaluronic acid may also be used, andthis may have the effect of promoting sustained duration in thecirculation. Other suitable means for the introduction of the desiredmolecule include implantable drug delivery devices.

In another aspect, pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

In another embodiment, a pharmaceutical composition may be formulatedfor inhalation. For example, a binding agent may be formulated as a drypowder for inhalation. Polypeptide or nucleic acid molecule inhalationsolutions may also be formulated with a propellant for aerosol delivery.In yet another embodiment, solutions may be nebulized. Pulmonaryadministration is further described in PCT Application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, binding agentmolecules that are administered in this fashion can be formulated withor without those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the binding agent molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

Pharmaceutical compositions for oral administration can also befornulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Another pharmaceutical composition may involve an effective quantity ofbinding agent in a mixture with non-toxic excipients that are suitablefor the manufacture of tablets. By dissolving the tablets in sterilewater, or other appropriate vehicle, solutions can be prepared in unitdose form. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving binding agent molecules insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. Seefor example, PCT/US93/00829 that describes controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. Additional examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate[Sidman et al., Biopolymers, 22:547-556 (1983)], poly(2-hydroxyethyl-methacrylate) [Langer et al., J. Biomed. Mater. Res.,15:167-277, (1981)] and [Langer et al., Chem. Tech., 12:98-105(1982)],ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., Proc. Natl.Acad. Sci. (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a sihgle-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which the bindingagent molecule is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.In other embodiments, the dosage may range from 0.1 mg/kg up to about100 mg/kg;, or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100mg/kg.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The exact dosage will be determined in light of factors related to thesubject requiring treatment. Dosage and administration are adjusted toprovide sufficient levels of the active compound or to maintain thedesired effect. Factors that may be taken into account include theseverity of the disease state, the general health of the subject, theage, weight, and gender of the subject, time and frequency ofadministration, drug combination(s), reaction sensitivities, andresponse to therapy. Long-acting pharmaceutical compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

The frequency of dosing will depend upon the pharmacokinetic parametersof the binding agent molecule in the formulation used. Typically, acomposition is administered until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as multiple doses (at the same or differentconcentrations/dosages) over time, or as a continuous infusion. Furtherrefinement of the appropriate dosage is routinely made. Appropriatedosages may be ascertained through use of appropriate dose-responsedata.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, urethral, vaginal, or rectalmeans, by sustained release systems or by implantation devices. Wheredesired, the compositions may be administered by bolus injection orcontinuously by infusion, or by implantation device.

Alternatively or additionally, the composition may be administeredlocally via implantation of a membrane, sponge, or another appropriatematerial on to which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

In some cases, it may be desirable to use pharmaceutical compositions inan ex vivo manner. In such instances, cells, tissues, or organs thathave been removed from the patient are exposed to the pharmaceuticalcompositions after which the cells, tissues and/or organs aresubsequently implanted back into the patient.

In other cases, a binding agent of the present invention such as apeptibody can be delivered by implanting certain cells that have beengenetically engineered, using methods such as those described herein, toexpress and secrete the polypeptide. Such cells may be animal or humancells, and may be autologous, heterologous, or xenogeneic. Optionally,the cells may be immortalized. In order to decrease the chance of animmunological response, the cells may be encapsulated to avoidinfiltration of surrounding tissues. The encapsulation materials aretypically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Combination Therapy

Specific binding agents of the invention (such as peptibodies) can beutilized in combination with other therapeutics in the treatment ofdiseases associated with Ang-2 expression. These other therapeuticsinclude, for example radiation treatment, chemotherapy, and targetedtherapies such as Herceptin™, Rituxan™, Gleevec™, and the like.Additional combination therapies not specifically listed herein are alsowithin the scope of the present invention.

The invention thus includes administration of one or more specificbinding agent of the invention administered to the same patient incombination with one or more additionally suitable agent(s), each beingadministered according to a regimen suitable for that medicament. Thisincludes concurrent administration of a specific binding agent of theinvention and one or more suitable agents. As used herein, the terms“concurrently administered” and “concurrent administration” encompasssubstantially simultaneous administration of one or more specificbinding agent (such as a peptide or peptibody) according to theinvention and one or more additionally suitable agents(s).

As used herein, the term, “non-concurrent” administration encompassesadministering one or more selective binding agent (such as a peptide orpeptibody) according to the invention and one or more additionallysuitable agent(s), at different times, in any order, whether overlappingor not. This includes, but is not limited to, sequential treatment (suchas pretreatment, post-treatment, or overlapping treatment) with thecomponents of the combination, as well as regimens in which the drugsare alternated, or wherein one component is administered long-term andthe other(s) are administered intermittently. Components may beadministered in the same or in separate compositions, and by the same ordifferent routes of administration.

Chemotherapy treatment can employ anti-neoplastic agents including, forexample, alkylating agents including: nitrogen mustards, such asmechlorethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemeiamine (TEM), triethylene,. thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; ppipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycinC, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platiniumcoordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as fiutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

Combination therapy with growth factors can include cytokines,lymphokines, growth factors, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cellfactor, and erythropoietin. Other are compositions can include knownangiopoietins, for example Ang-1, -2, -4, -Y, and/or the human Ang-likepolypeptide, and/or vascular endothelial growth factor (VEGF). Growthfactors include angiogenin, bone morphogenic protein-1, bone morphogenicprotein-2, bone morphogenic protein-3, bone morphogenic protein-4, bonemorphogenic protein-5, bone morphogenic protein-6, bone morphogenicprotein-7, bone morphogenic protein-8, bone morphogenic protein-9, bonemorphogenic protein-10, bone morphogenic protein-11, bone morphogenicprotein-12, bone morphogenic protein-13, bone morphogenic protein-14,bone morphogenic protein-15, bone morphogenic protein receptor IA, bonemorphogenic protein receptor IB, brain derived neurotrophic factor,ciliary neutrophic factor, ciliary neutrophic factor receptor,cytokine-induced neutrophil chemotactic factor 1, cytokine-inducedneutrophil, chemotactic factor 2, cytokine-induced neutrophilchemotactic factor 2, endothelial cell growth factor, endothelin 1,epidermal growth factor, epithelial-derived neutrophil attractant,fibroblast growth factor 4, fibroblast growth factor 5, fibroblastgrowth factor 6, fibroblast growth factor 7, fibroblast growth factor 8,fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblastgrowth factor 9, fibroblast growth factor 10, fibroblast growth factoracidic, fibroblast growth factor basic, glial cell line-derivedneutrophic factor receptor-1, glial cell line-derived neutrophic factorreceptor-2, growth related protein, growth related protein-1, growthrelated protein-2, growth related protein-3, heparin binding epidermalgrowth factor, hepatocyte growth factor, hepatocyte growth factorreceptor, insulin-like growth factor I, insulin-like growth factorreceptor, insulin-like growth factor II, insulin-like growth factorbinding protein, keratinocyte growth factor, leukernia inhibitoryfactor, leukernia inhibitory factor receptor-1, nerve growth factornerve growth factor receptor, neurotrophin-3, neurotrophin-4, placentagrowth factor, placenta growth factor 2, platelet-derived endothelialcell growth factor, platelet derived growth factor, platelet derivedgrowth factor A chain, platelet derived growth factor AA, plateletderived growth factor AB, platelet derived growth factor B chain,platelet derived growth factor BB, platelet derived growth factorreceptor-1, platelet derived growth factor receptor-2, pre-B cell growthstimulating factor, stem cell factor, stem cell factor receptor,transforming growth factor-1, transforming growth factor-2, transforminggrowth factor-1, transforming growth factor-1.2, transforming growthfactor-2, transforming growth factor-3, transforming growth factor-5,latent transforming growth factor-1, transforming growth factor-1binding protein I, transforming growth factor-1 binding protein II,transforming growth factor-1 binding protein III, tumor necrosis factorreceptor type I (TNF-R1), tumor necrosis factor receptor type II(TNF-R2), urokinase-type plasminogen activator receptor, vascularendothelial growth factor, and chimeric proteins and biologically orimmunologically active fragments thereof.

It will be appreciated that the specific binding agents (such aspeptides or peptibodies) of the invention may be administered with oneor more anti-inflammatory agents. As used herein, the term“anti-inflammatory agent” refers generally to any agent that reducesinflammation or swelling in a patient. A number of exemplaryanti-inflammatory agents are recited herein, but it will be appreciatedthat there may be additional suitable anti-inflammatory agents notspecifically recited herein, but which are encompassed by the presentinvention.

The anti-inflammatory agent can be, for example, a compound thatinhibits the interaction of inflammatory cytokines with their receptors.Examples of cytokine inhibitors useful in combination with the specificbinding agents of the invention include, for example, antagonists (suchas antibodies) of TGF-β, as well as antagonists (such as antibodies)directed against interleukins involved in inflammation. Suchinterleukins are described herein and preferably include, but are notlimited to, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12,IL-13, IL-17, and IL-18. See Feghali, et al., Frontiers in Biosci.,2:12-26 (1997).

Specific binding agents of the invention also may be administered incombination with inhibitors of Protein Kinase A Type 1 to enhance T cellproliferation in HIV-infected patients who are receiving anti-retroviraltherapy.

Nerve growth factors (NGFs) also can be combined with the specificbinding agents of the invention to treat certain conditions. Suchconditions include neurodegenerative diseases, spinal cord injury andmultiple sclerosis. Other conditions treatable with this combination areglaucoma and diabetes.

A preferred combination therapy relates to a specific binding agent ofthe invention administered to a patient in combination with one or moresuitable IL-1 inhibitor. Inhibitors of IL-1 include, but are not limitedto, receptor-binding peptide fragments of IL-1, antibodies directedagainst IL-1 or IL-1 beta or IL-1 receptor type I, and recombinantproteins comprising all or portions of receptors for IL-1 or modifiedvariants thereof, including genetically-modified muteins, multimericforms and sustained-release formulations. Specific antagonists includeIL-1ra polypeptides, IL-1 beta converting enzyme (ICE) inhibitors,antagonistic type I IL-1 receptor antibodies, IL-1 binding forms of typeI IL-1 receptor and type H IL-1 receptor, antibodies to IL-1, includingIL-1 alpha and IL-1 beta and other IL-1 family members, and atherapeutic known as IL-1 Trap (Regeneron). IL-1ra polypeptides includethe forms of IL-1ra described in U.S. Pat. No. 5,075,222 and modifiedforms and variants including those described in U.S. Pat. No. 5,922,573,WO 91/17184, WO 92 16221, and WO 96 09323. IL-1 beta converting enzyme(ICE) inhibitors include peptidyl and small molecule ICE inhibitorsincluding those described in PCT patent applications WO 91/15577; WO93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; and European patentapplication 0 547 699. Non-peptidyl compounds include those described inPCT patent application WO 95/26958, U.S. Pat. Nos. 5,552,400, 6,121,266,and Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996). AdditionalICE inhibitors are described in U.S. Pat. Nos. 6,162,790, 6,204,261,6,136,787, 6,103,711, 6,025,147, 6,008,217, 5,973,111, 5,874,424,5,847,135, 5,843,904, 5,756,466, 5,656,627, 5,716,929. IL-1 bindingforms of Type I IL-1 receptor and type II IL-1 receptor are described inU.S. Pat. Nos. 4,968,607, 4,968,607, 5,081,228, Re 35,450, 5,319,071,and 5,350,683. Other suitable IL-1 antagonists include, but are notlimited to, peptides derived from IL-1 that are capable of bindingcompetitively to the IL-1 signaling receptor, IL-1 R type I. Additionalguidance regarding certain IL-1 (and other cytokine) antagonists can befound in U.S. Pat. No. 6,472,179.

Additionally, TNF inhibitors are suitable, and include, but are notlimited to, receptor-binding peptide fragments of TNFα, antisenseoligonucleotides or ribozymes that inhibit TNFα production, antibodiesdirected against TNFα, and recombinant proteins comprising all orportions of receptors for TNFα or modified variants thereof, includinggenetically-modified muteins, multimeric forms and sustained-releaseformulations. Also suitable are TACE (Tumor Necrosis Factor-α ConvertingEnzyme) inhibitors, such as TAPI (Immunex Corp.) and GW-3333X (GlaxoWellcome Inc.). Also suitable are molecules that inhibit the formationof the IgA-α₁AT complex, such as the peptides disclosed in EP 0 614 464B, or antibodies against this complex. Additionally suitable moleculesinclude, but are not limited to, TNFα-inhibiting disaccharides, sulfatedderivatives of glucosamine, or other similar carbohydrates described inU.S. Pat. No. 6,020,323. Further suitable molecules include peptide TNFαinhibitors disclosed in U.S. Pat. Nos. 5,641,751 and 5,519,000, and theD-amino acid-containing peptides described in U.S. Pat. No. 5,753,628.In addition, inhibitors of TNFα converting enzyme are also suitable. WO01/03719 describes further additional agents which can be used incombination in accordance with the invention.

Still Further suitable compounds include, but are not limited to, smallmolecules such as thalidomide or thalidomide analogs, pentoxifylline, ormatrix metalloproteinase (MMP) inhibitors or other small molecules.Suitable MMP inhibitors for this purpose include, for example, thosedescribed in U.S. Pat. Nos. 5,883,131, 5,863,949 and 5,861,510 as wellas mercapto alkyl peptidyl compounds as described in U.S. Pat. No.5,872,146. Other small molecules capable of reducing TNFα production,include, for example, the molecules described in U.S. Pat. Nos.5,508,300, 5,596,013, and 5,563,143. Additional suitable small moleculesinclude. but are not limited to, MMP inhibitors as described in U.S.Pat. Nos. 5,747,514, and 5,691,382, as well as hydroxamic acidderivatives such as those described in U.S. Pat. No. 5,821,262. Furthersuitable molecules include, for example, small molecules that inhibitphosphodiesterase IV and TNFα production, such as substituted oximederivatives (WO 96/00215), quinoline sulfonamides (U.S. Pat. No.5,834,485), aryl furan derivatives (WO 99/18095) and heterobicyclicderivatives (WO 96/01825; GB 2 291 422 A). Also useful are thiazolederivatives that suppress TNFα and IFNγ (WO 99/15524), as well asxanthine derivatives that suppress TNFα and other proinflammatorycytokines (see, for example, U.S. Pat. Nos. 5,118,500, 5,096,906 and5,196,430). Additional small molecules useful for treating thehereindescribed conditions include those disclosed in U.S. Pat. No.5,547,979.

Further examples of drugs and drug types which can be administered bycombination therapy include, but are not limited to, antivirals,antibiotics, analgesics (e.g., acetaminophen, codeine, propoxyphenenapsylate, oxycodone hydrochloride, hydrocodone bitartrate, tramadol),corticosteroids, antagonists of inflammatory cytokines,Disease-Modifying Anti-Rheumatic Drugs (DMARDs), Non-SteroidalAnti-Inflammatory drugs (NSAIDs), and Slow-Acting Anti-Rheumatic Drugs(SAARDs).

Exemplary Disease-Modifying Anti-Rheumatic Drugs (DMARDs) include, butare not limited to: Rheumatrex™ (methotrexate); Enbrel® (etanercept);Remicade® (infliximab); Humira™ (adalimumab); Segard® (afelimomab);Arava™ (leflunomide); Kineret™ (anakinra); Arava™ (leflunomide);D-penicillamine; Myochrysine; Plaquenil; Ridaura™ (auranofin); Solganal;lenercept (Hoffman-La Roche); CDP870 (Celltech); CDP571 (Celltech), aswell as the antibodies described in EP 0 516 785 B1, U.S. Pat. No.5,656,272, EP 0 492 448 A1; onercept (Serono; CAS reg. no. 199685-57-9);MRA (Chugai); Imuran™ (azathioprine); NFKB inhibitors; Cytoxan™(cyclophosphamide); cyclosporine; hydroxychloroquine sulfate;minocycline; sulfasalazine; and gold compounds such as oral gold, goldsodium thiomalate and aurothioglucose.

Further suitable molecules include, for example, soluble TNFRs derivedfrom the extracellular regions of TNFα receptor molecules other than thep55 and p75 TNFRs, such as for example the TNFR described in WO99/04001, including TNFR-Ig's derived from this TNFR. Additionalsuitable TNFα inhibitors are suitable for use as described herein. Theseinclude the use not only of an antibody against TNFα or TNFR asdescribed herein, but also a TNFα-derived peptide that can act as acompetitive inhibitor of TNFα (such as those described in U.S. Pat. No.5,795,859 or U.S. Pat. No. 6,107,273), TNFR-IgG fusion proteins, such asone containing the extracellular portion of the p55 TNFα receptor, asoluble TNFR other than an IgG fusion protein, or other molecules thatreduce endogenous TNFα levels, such as inhibitors of the TNFα convertingenzyme (see e.g., U.S. Pat. No. 5,594,106), or small molecules or TNFαinhibitors, a number of which are described herein.

With respect to antibodies to TNF, although dose will optimally bedetermined by an experienced healthcare provider in accordance with thespecific needs of the patient in mind, one exemplary preferred doserange for an antibody against TNFα is 0.1 to 20 mg/kg, and morepreferably is 1-10 mg/kg. Another preferred dose range for anti-TNFαantibody is 0.75 to 7.5 mg/kg of body weight.

The present invention can also utilize a specific binding agent and anyof one or more Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDsowe their anti-inflammatory action, at least in part, to the inhibitionof prostaglandin synthesis. Goodman and Gilman, The PharmacologicalBasis of Therapeutics, MacMillan 7th Edition (1985). NSAIDs can becharacterized into nine groups: (1) salicylic acid derivatives; (2)propionic acid derivatives; (3) acetic acid derivatives; (4) fenamicacid derivatives; (5) carboxylic acid derivatives; (6) butyric acidderivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones. Examples ofNSAIDs include, but are not limited to: Anaprox™, Anaprox DS™ (naproxensodium); Ansaid™ (flurbiprofen); Arthrotec™ (diclofenacsodium+misoprostil); Cataflam™/Voltaren™ (diclofenac potassium);Clinoril™ (sulindac); Daypro™ (oxaprozin); Disalcid™ (salsalate);Dolobid™ (diflunisal); EC Naprosyn™ (naproxen sodium); Feldene™(piroxicam); Indocin™, Indocin SR™ (indomethacin); Lodine™ , Lodine XL™(etodolac); Motrin™ (ibuprofen); Naprelan™ (naproxen); Naprosyn™(naproxen); Orudis™ , (ketoprofen); Oruvail™ (ketoprofen); Relafen™(nabumetone); Tolectin™ , (tolmetin sodium); Trilisate™ (cholinemagnesium trisalicylate); Cox-1 inhibitors; Cox-2 Inhibitors such asVioxx™ (rofecoxib); Arcoxia™ (etoricoxib), Celebrex™ (celecoxib); Mobic™(meloxicam); Bextra™ (valdecoxib), Dynastat™ paracoxib sodium; Prexige™(lumiracoxib), and nambumetone. Additional suitable NSAIDs, include, butare not limited to, the following: ε-acetamidocaproic acid,S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,anitrazafen, antrafenine, bendazac, bendazac lysinate, benzydamine,beprozin, broperamole, bucolome, bufezolac, ciproquazone, cloximate,dazidamine, deboxamet, detomidine, difenpiramide, difenpyramide,difisalamine, ditazol, emorfazone, fanetizole mesylate, fenflumizole,floctafenine, flumizole, flunixin, fluproquazone, fopirtoline, fosfosal,guaimesal, guaiazolene, isonixim, lefetamine HCl, leflunomide,lofemizole, lotifazole, lysin clonixinate, meseclazone, nabumetone,nictindole, nimesulide, orgotein, orpanoxin, oxaceprolm, oxapadol,paranyline, perisoxal, perisoxal citrate, pifoxime, piproxen, pirazolac,pirfenidone, proquazone, proxazole, thielavin B, tiflamizole,timegadine, tolectin, tolpadol, tryptamid and those designated bycompany code number such as 480156S, AA861, AD1590, AFP802, AFP860,A177B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EB382, EL508,F1044, FK-506, GV3658, ITF182, KCNTE16090, KME4, LA2851, MR714, MR897,MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440,SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901(4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770.Structurally related NSAIDs having similar analgesic andanti-inflammatory properties to the NSAIDs are also encompassed by thisgroup.

Suitable SAARDs or DMARDS include, but are not limited to: allocupreidesodium, auranofin, aurothioglucose, aurothioglycanide, azathioprine,brequinar sodium, bucillamine, calcium3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine,clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone,15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquinegold salt, gold sodium thiomalate, gold sodium thiosulfate),hydroxychloroquine, hydroxyurea, kebuzone, levamisole, lobenzarit,melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolatemofetil, myoral, nitrogen mustard, D-penicillamine, pyridinol imidazolessuch as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin andvincristine. Structurally related SAARDs or DMARDs having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

Inhibitors of kinases in signaling cascades are also suitable agents forcombination with the specific binding agents of the invention. Theseinclude, but are not limited to, agents which are capable of inhibitingP-38 (a.k.a., “RK” or “SAPK-2”, Lee et al., Nature, 372:739 (1994). P-38is described as a serine/threonine kinase (see Han et al., BiochimicaBiophysica Acta, 1265:224-227 (1995). Inhibitors of P-38 have been shownto intervene between the extracellular stimulus and the secretion ofIL-1 and TNFα from the cell involves blocking signal transductionthrough inhibition of a kinase which lies on the signal pathway.

Additionally suitable are MK2 inhibitors, and tpl-2 inhibitors.Additionally, T-cell inhibitors are also suitable, including, forexample, ctla4, CsA, Fk-506, OX40, OX40R-Fc, OX40 antibody, OX40 ligand,OX40 ligand antibody, lck, and ZAP70. Also suitable are retinoids,including oral retinoids, as well as antagonists of TGF-β.

Further suitable agents for combination with the specific binding agentsof the invention include, for example, any of one or more salicylic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. Such salicylic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: acetaminosalol,aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate diflusinal,etersalate, fendosal, gentisic acid, glycol salicylate, imidazolesalicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate,1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate,phenyl salicylate, salacetamide, salicylamide O-acetic acid, salsalateand sulfasalazine. Structurally related salicylic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group. Additionally suitable agentsinclude, for example propionic acid derivatives, prodrug esters orpharmaceutically acceptable salts thereof. The propionic acidderivatives, prodrug esters and pharmaceutically acceptable saltsthereof comprise: alminoprofen, benoxaprofen, bucloxic acid, carprofen,dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen,furcloprofen, ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen,isoprofen, ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin,piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid,pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen. Structurallyrelated propionic acid derivatives having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup. Also suitable for use are acetic acid derivatives, prodrug estersor pharmaceutically acceptable salts thereof. The acetic acidderivatives, prodrug esters and pharmaceutically acceptable saltsthereof comprise: acemetacin, alclofenac, amfenac, bufexamac,cinmetacin, clopirac, delmetacin, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, zidometacin andzomepirac. Structurally related acetic acid derivatives having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group. Further suitable for use as described hereinare fenamic acid derivatives, prodrug esters or pharmaceuticallyacceptable salts thereof. The fenamic acid derivatives, prodrug estersand pharmaceutically acceptable salts thereof comprise: enfenamic acid,etofenarnate, flufenamic acid, isonixin, meclofenamic acid,meclofenamate sodium, medofenamic acid, mefanamic acid, niflumic acid,talniflumate, terofenamate, tolfenamic acid and ufenamate. Structurallyrelated fenamic acid derivatives having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

Also suitable are carboxylic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof which can be used comprise:clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine.Structurally related carboxylic acid derivatives having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group. Additionally suitable are butyric acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The butyric acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group. Oxicams, prodrugesters or pharmaceutically acceptable salts thereof are also suitable.Oxicams, prodrug esters and pharmaceutically acceptable salts thereofcomprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicamand 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide.Structurally related oxicams having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup. Pyrazoles, prodrug esters or pharmaceutically acceptable saltsthereof are also suitable. The pyrazoles, prodrug esters andpharmaceutically acceptable salts thereof which may be used comprise:difenamizole and epirizole. Structurally related pyrazoles havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group. Furthermore, pyrazolones, prodrug estersor pharmaceutically acceptable salts thereof are suitable. Thepyrazolones, prodrug esters and pharmaceutically acceptable saltsthereof which may be used comprise: apazone, azapropazone,benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone,phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzoneand thiazolinobutazone. Structurally related pyrazalones having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

Also suitable are prodrug esters or pharmaceutically acceptable saltsthereof for the treatment of TNF-mediated diseases. Corticosteroids,.prodrug esters and pharmaceutically acceptable salts thereof includehydrocortisone and compounds which are derived from hydrocortisone, suchas 21-acetoxy-pregnenolone, alclomerasone, algestone, amcinonide,beclomethasone, beta-methasone, betamethasone valerate, budesonide,chloroprednisone, clobetasol, clobetasol propionate, clobetasone,clobetasone butyrate, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacon, desonide, desoximerasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flumethasone pivalate,flunisolide, flucinolone acetonide, fluocinonide, fluorocinoloneacetonide, fluocortin butyl, fluocortolone, fluorocortolone hexanoate,diflucortolone valerate, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandenolide, formocortal,halcinonide, halometasone, halopredone acetate, hydrocortamate,hydrocortisone, hydrocortisone acetate, hydro-cortisone butyrate,hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednicolone, mometasone furoate, paramethasone,-prednicarbate,prednisolone,-prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

Antimicrobials (and prodrug esters or pharmaceutically acceptable saltsthereof) are also suitable for combination use as described herein.Suitable antimicrobials include, for example, ampicillin, amoxycillin,aureomicin, bacitracin, ceftazidime, ceftriaxone, cefotaxime,cephachlor, cephalexin, cephradine, ciprofloxacin, clavulanic acid,cloxacillin, dicloxacillan, erythromycin, flucloxacillan, gentamicin,gramicidin, methicillan, neomycin, oxacillan, penicillin and vancomycin.Structurally related antimicrobials having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

Additional suitable compounds include, but are not limited to: BN 50730;tenidap; E 5531; tiapafant PCA 4248; nimesulide; panavir; rolipram; RP73401; peptide T; MDL 201,449A;(1R,3S)-Cis-1-[9-(2,6-diaminopurinyl)]-3-hydroxy4-cyclopentenehydrochloride;(1R,3R)-trans-1-[9-(2,6-diamino)purine]-3-acetoxycyclopentane; (1R,3R)-trans-1-(9-adenyl)-3-azidocyclopentane hydrochloride and(1R,3R)-trans-1-[6-hydroxy-purin-9-yl)-3-azidocyclopentane.

It has been found that IL4 can induce an inflammatory effect in someinstances, such as in asthma, in which over-expression of IL-4 in thelungs causes epithelial cell hypertrophy and an accumulation oflymphocytes, eosinophils, and neutrophils. This response isrepresentative of the main features of the proinflammatory responseinduced by other Th2 cytokines. As noted above, therefore, inhibitors ofIL4 are also useful in accordance with the invention. Additionally, itwill be appreciated that certain immunosuppressant drugs can also beused in the treatment of arthritis, including, but not limited to, iNOSinhibitors, and 5-lipoxygenase inhibitors.

Ginger has been shown to have certain anti-inflammatory properties, andis therefore suitable for use as an anti-inflammatory agent inaccordance with the invention, as is chondroitin.

The above listings are by way of example only, and do not preclude theuse of other compounds or treatments which can be used concurrently withthe compounds described herein that are known by those skilled in theart or that could be arrived at by those skilled in the art using theguidelines set forth in this specification.

Immunotherapeutics

Immunotherapeutics generally rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectorsmay be, for example, a peptibody of the present invention thatrecognizes some marker on the surface of a target cell. The peptibodyalone may serve as an effector of therapy or it may recruit other cellsto actually effect cell killing. The peptibody may also be conjugated toa drug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and thus may merely serve as a targetingagent.

According to the present invention, mutant forms of Ang-2 may betargeted by immunotherapy either peptibodies or peptibody conjugates ofthe invention. It is particularly contemplated that the peptibodycompositions of the invention may be used in a combined therapy approachin conjunction with Ang-2 targeted therapy.

Passive immunotherapy has proved to be particularly effective against anumber of cancers. See, for example, WO 98/39027.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLE 1 Ang-2 Expression in Pathological and Normal Tissue

Ang-2 expression was examined in normal and pathological tissue using insitu hybridization. Fragments of the human (Genbank Accession Number:AF004327, nucleotides 1274-1726) and murine (Genbank Accession Number:AF004326, nucleotides 1135-1588) Ang-2 sequences were amplified byreverse transcriptase-PCR from human or murine fetal lung cDNA, clonedinto the pGEM-T plasmid and verified by sequencing. ³³P-labeledantisense RNA probes were transcribed from linearized plasmid templatesusing ³³P-UTP and RNA polymerase. Blocks of formaldehyde-fixed,paraffin-embedded tissues were sectioned at 5 μm and collected oncharged slides. Prior to in situ hybridization, tissues werepermeabilized with 0.2 M HCL, followed by digestion with Proteinase K,and acetylation with triethanolamine and acetic anhydride. Sections werehybridized with the radio labeled probe overnight at 55° C. thensubjected to RNase digestion and a high stringency wash in about 0.1×SSCat 55° C. Slides were dipped in Kodak NTB2 emulsion, exposed at 4° C.for 2-3 weeks, developed, and counterstained. Sections were examinedwith dark field and standard illumination to allow simultaneousevaluation of tissue morphology and hybridization signal.

The results indicated that in the normal postnatal human, Ang-2expression is restricted to the few tissues containing angiogenicvasculature, such as the ovary, placenta, and uterus. No Ang-2expression was detectable in normal adult human heart, brain, kidney,liver, lung, pancreas, spleen, muscle, tonsil, thymus, appendix, lymphnode, gall bladder, prostate or testis. In five-week-old mouse (but notadult monkey or human), kidneys displayed prominent Ang-2 expression inthe vasa recta. To determine whether this expression was a remnant ofembryonic development, this experiment was repeated on kidneys derivedfrom mice ranging in age up to one-year-old using the murine Ang-2 probeand conditions described above. Ang-2 expression was observed todecrease during neonatal development, but was still evident in kidneysof one-year-old mice.

Ang-2 expression was also detected in virtually all tumor types tested,including, primary human tumors such as colon carcinoma (5 cases),breast carcinoma (10 cases), lung carcinoma (8 cases), glioblastoma (1case), metastatic human tumors such as breast carcinoma (2 cases), lungcarcinoma (2 cases) and ovarian carcinoma (2 cases) which had metastizedto brain, and rodent tumor models such as C6 (rat glioma), HT29 (humancolon carcinoma), Colo-205 (human colon carcinoma), HCT116 (human coloncarcinoma), A431 (human epidermoid carcinoma), A673 (humanrhabdomyosarcoma), HT1080 (human fibrosarcoma), PC-3 (human prostatecarcinoma), B16F10 (murine melanoma), MethA (murine sarcoma), and Lewislung carcinoma mets. Additionally, Ang-2 expression was detected inneovessels growing into a Matrigel plug in response to VEGF and in amouse hypoxia model of retinopathy of prematurity.

EXAMPLE 2 Molecular Assays to Evaluate Ang-2 Peptibodies

Molecular assays (Affinity ELISA, Neutralization ELISA, and BIAcore)were developed to assess direct peptibody binding to Ang-2 and relatedfamily members, and the effect of peptibodies on the Ang-2:Tie-2interaction. These in vitro assays are described as follows.

Affinity ELISA

For the initial screening of candidate anti-Ang-2 peptibodies, purifiedhuman Ang-2 (R&D Systems, Inc; catalog number 623-AN; Ang-2 is providedas a mixture of 2 truncated versions) or murine Ang-2 polypeptide(prepared as described above) were used. For confirmatory bindingassays, human Ang-2 was obtained from conditioned media of human 293Tcells transfected with full length human Ang-2 DNA and cultured in serumfree Dulbecco's Modified Eagle Medium (DMEM) containing about 50micrograms per ml of bovine serum albumin (BSA).

Using microtiter plates, approximately 100 microliters per well of Ang-2was added to each well and the plates were incubated about 2 hours,after which the plates were washed with phosphate buffered saline (PBS)containing about 0.1 percent Tween-20 four times. The wells were thenblocked using about 250 microliters per well of about 5 percent BSA inPBS, and the plates were incubated at room temperature for about 2hours. After incubation, excess blocking solution was discarded, andabout 100 microliters of each candidate anti-Ang-2 peptibody was addedto a well in a dilution series starting at a concentration of about 40nanomolar and then serially diluting 4-fold in PBS containing about 1percent BSA. The plates were then incubated overnight at roomtemperature. After incubation, plates were washed with PBS containingabout 0.1 percent Tween-20. Washing was repeated four additional times,after which about 100 microliters per well of goat anti-humanIgG(Fc)-HRP (Pierce Chemical Co., catalog #31416) previously diluted1:5000 in PBS containing 1 percent BSA was added. Plated were incubatedapproximately 1 hour at room temperature. Plates were then washed fivetimes in PBS containing about 0.1 percent Tween-20, after which about100 microliters per well of TMB (3,3′,5,5′-Tetramethylbenzidine LiquidSubstrate System; Sigma Chemical Company, St. Louis, Mo., catalog numberT8665) substrate was added and plates were incubated about 5-15 minutesuntil blue color developed. Absorbance was then read in aspectrophotometer at about 370 nm.

Neutralization ELISA

Microtiter plates to which human Ang-2 polypeptide was bound wereprepared as described for the Affinity ELISA. Candidate anti-Ang-2peptibodies were titrated from 1000nM to 0.2pM in 4-fold dilutions in asolution of PBS containing about 1% BSA and about 1 nM Tie-2 (providedas a Tie-2-Fc molecule where the Tie-2 portion contains only the solubleextracellular portion of the molecule; R&D Systems, catalog number313-TI). After about 100 microliters of the antibody/Tie-2 solution wasadded to each well, the plates were incubated overnight at roomtemperature, and then washed five times in PBS containing about 0.1percent Tween-20. After washing, about 100 microliters per well ofanti-Tie-2 antibody (Pharmingen Inc., catalog #557039) was added to afinal concentration of about 1 microgram per ml, and the plates wereincubated about 1 hour at room temperature. Next, about 100 microlitersper well of goat anti-mouse-IgG-HRP (Pierce Chemical CO., catalog#31432) was added at a dilution of 1:10,000 in PBS containing about 1percent BSA. Plates were incubated at room temperature for about 1 hour,after which they were washed five times with PBS containing about 0.1percent Tween-20. About 100 microliters per well of TMB substrate(described above) was then added and color was allowed to develop.Absorbance was then read in a spectrophotomer at 370 nm.

Affinity BIAcore

An affinity analysis of each candidate Ang-2 peptibody was performed ona BIAcore2000 (Biacore, Inc., Piscataway, N.J.) with PBS and 0.005percent P20 surfactant (Biacore, Inc.) as running buffer. RecombinantProtein G (Repligen, Needham, Mass.) was immobilized to a research gradeCM5 sensor chip (Biacore, Inc.) via primary amine groups using the AmineCoupling Kit (Biacore, Inc.) according to the manufacturer's suggestedprotocol.

Binding assays were carried out by first capturing about 100 Ru of eachcandidate anti-Ang-2 peptibody to the immobilized Protein G, after whichvarious concentrations (0-100 nM) of huAng-2 or mAng-2 were injectedover the bound antibody surface at a flow rate of 50 μl/min for 3minutes. Peptibody binding kinetic parameters including k_(a)(association rate constant), k_(d) (dissociation rate constant) andK_(D) (dissociation equilibrium constant) were determined using the BIAevaluation 3.1 computer program (Biacore, Inc.). Lower dissociationequilibrium constants indicated greater affinity of the peptibody forAng-2.

EXAMPLE 3 Identification of Ang-2 Binding Peptides

1. Ang-2-Coated Magnetic Bead Preparation

A. Ang-2 Immobilization on Magnetic Beads

For non-specific elution, the biotinylated Ang-2 protein (BiotinylatedRecombinant Human Angiopoietin-2, R&D Systems, Inc.; catalog number BT623) was immobilized on the Streptavidin Dynabeads (Dynal, Lake Success,N.Y.) at a concentration of about 4 μg of the biotinylated Ang-2 proteinper 100 μl of the bead stock from the manufacturer for all three roundsof selection. For antigen (Ang-2) and receptor (Tie-2) elutions, 2 μg ofbiotinylated Ang-2 protein was immobilized on 50 μl of the StreptavidinDynabeads for the second rounds of selection. The coating concentrationwas reduced to about 1 μg of biotinylated Ang-2 protein per 50 μg of thebead stock for the third round of selection. By drawing the beads to oneside of a tube using a magnet and pipetting away the liquid, the beadswere washed five times with the phosphate buffer saline (PBS) andresuspended in PBS. The biotinylated Ang-2 protein was added to thewashed beads at the above concentration and incubated with rotation for1 hour at room temperature, followed by a few hours to an overnightincubation at 4° C. with rotation. Ang-2-coated beads were then blockedby adding BSA to about 1% final concentration and incubating overnightat 4° C. with rotation. The resulting Ang-2 coated beads were thenwashed five times with PBS before being subjected to the selectionprocedures.

B. Negative Selection Bead Preparation

Additional beads were also prepared for negative selections. For eachpanning condition, 500 μl of the bead stock from the manufacturer wassubjected to the above procedure (section 1A) except that the incubationstep with biotinylated Ang-2 was omitted. In the last washing step, thebeads were divided into five 100 μl aliquots.

2. Selection of Ang-2 Binding Phage

A. Overall Strategy

Three filamentous phage libraries, designated as “TN8-IX” (5×10⁹independent transformants), “TN12-I” (1.4×⁹ independent transformants),and “Linear” (2.3×⁹ independent transformants) (all from Dyax Corp.),were used to select for Ang-2 binding phage. Each library was thensubjected to either non-specific elution, Ang-2 elution, and receptorelution (Tie-2). Nine different panning conditions were carried out forAng-2 (TN8-IX using the non-specific elution method, TN8-IX using theAng-2 elution method, TN8-IX using the Tie-2 elution method, TN12-Iusing the non-specific elution method, TN12-I using the Ang-2 elutionmethod, and TN12-I using the Tie-2 elution method, Linear using thenon-specific elution method, Linear using the Ang-2 elution method, andLinear using the Tie-2 elution method). For all three libraries, thephage from the first round of selection were eluted only in anon-specific manner for further rounds of selection. The Ang-2 and Tie-2elutions were used in the second and third rounds of selection. For theLinear library, the selection was carried to only the second round forthe Ang-2 and Tie-2 elutions.

B. Negative Selection

For each panning condition, about 100 random library equivalents forTN8-IX and TN12-I libraries (about 5×10¹¹ pfu for TN8-IX, and about1.4×10¹¹ pfu for TN12-I) and about 10 random library equivalents for thelinear library (about 1×10¹¹ pfu) were aliquoted from the library stockand diluted to about 400 μl with PBST (PBS with 0.05% Tween-20). Afterthe last washing, liquid was drawn out from the first 100 μl aliquot ofthe beads prepared for negative selection (section 1B), theapproximately 400 μl diluted library stock was added to the beads. Theresulting mixture was incubated for about 10 minutes at room temperaturewith rotation. The phage supernatant was drawn out using the magnet andadded to the second 100 μl aliquot for another negative selection step.In this way, five negative selection steps were performed.

C. Selection Using the Ang-2 Protein Coated Beads

The phage supernatant after the last negative selection step (section1B) was added to the Ang-2 coated beads (section 1A). This mixture wasincubated with rotation for one to two hours at room temperature,allowing phage to bind to the target protein. After the supernatant wasdiscarded, the beads were washed about ten times with PBST followed bytwo washes with PBS.

D. Non-specific Elution

After the final washing liquid was drawn out (section 2C), about 1 ml ofMin A salts solution (60 mM K₂HPO₄, 33 mM KH₂PO₄, 7.6 mM (NH4)SO₄, and1.7 mM sodium citrate) was added to the beads. This bead mixture wasadded directly to a concentrated bacteria sample for infection (seebelow section 3A and 3B).

E. Antigen (Ang-2) Elution of Bound Phage

For round 2, after the last washing step (section 2C), the bound phagewere eluted from the magnetic beads by adding 100 μl of 1 pM, 0.1 nM,and 10 nM recombinant Ang-2 protein (Recombinant Human Angiopoietin-2,R&D Systems, Inc., Minneapolis, Minn.) successively with a 30-minuteincubation for each condition. The remaining phage were elutednon-specifically (section 2D). The eluted phage from 10 nM andnon-specific elutions were combined, and they were subjected to thethird round of selection (see Section 4, below).

For round 3, after the last washing step (section 2C), the bound phagewere eluted from the magnetic beads by adding about 1 nM recombinantAng-2 protein, and 10 nM recombinant Ang-2 protein successively with a30-minute incubation for each condition. In addition, the phage wereeluted with 1 ml of 100 mM triethylamine solution (Sigma, St. Louis,Mo.) for about 10 minutes on a rotator. The pH of the phage-containingthe triethylamine solution was neutralized with 0.5 ml of 1 M Tris-HCl(pH 7.5). After the last elution with 100 mM triethylamine solution, theremaining phage were eluted by adding beads to the bacteria (section2D).

F. Receptor (Tie-2) Elution of Bound Phage

For round 2, after the last washing step (section 2C), the bound phagewere eluted from the magnetic beads by adding about 100 μl of 1 pM, 0.1nM, and 10 nM recombinant Tie-2 protein (Recombinant Human Tie-2-FcChimera, R&D Systems, Inc., Minneapolis, Minn.) successively with a30-minute incubation for each condition. The remaining phage were elutednon-specifically (section 2D). The eluted phage from 10 nM andnon-specific elutions were combined and they were subjected to the thirdround of selection (see below section 4).

For round 3, after the last washing step (section 2C), the bound phagewere eluted from the magnetic beads by adding about 1 nM of recombinantAng-2 protein, and 10 nM recombinant Tie-2 protein successively with a30-minute incubation for each condition. In addition, the phage wereeluted with 1 ml of 100 mM triethylamine solution (Sigma, St. Louis,Mo.) for 10 minutes on a rotator. The pH of the phage containing thetriethylamine solution was neutralized with 0.5 ml of 1 M Tris-HCl (pH7.5). After the last elution with 100 mM triethylamine solution, theremaining phage were eluted by adding beads to the bacteria (section2D).

3. Amplification

A. Preparation of Plating Cells

Fresh E. Coli. (XL-1 Blue MRF′) culture was grown to an OD₆₀₀ of about0.5 in LB media containing about 12.5 μg/ml tetracycline. For eachpanning condition, about 20 ml of this culture was chilled on ice andcentrifuged. The bacteria pellet was resuspended in about 1 ml of theMin A Salts solution.

B. Transduction

Each mixture from each different elution method set forth above(sections 2D, 2E and 2F) was added to a concentrated bacteria sample(section 3A) and incubated at about 37° C. for about 15 minutes.Approximately 2 ml of NZCYM media (2XNZCYM, 50 μg/ml Ampicillin) wasadded to each mixture and incubated at about 37° C. for 15 minutes. Theresulting 4 ml solution was plated on a large NZCYM agar platecontaining about 50 μg/ml Ampicillin and incubated overnight at 37° C.

C. Phage Harvesting

Each bacteria/phage mixture was grown overnight on a large NZCYM agarplate (section 3B), after which they were scraped off into about 35 mlof LB media. The agar plate was further rinsed with additional 35 ml ofLB media. The resulting bacteria/phage mixture in LB media wascentrifuged to pellet the bacteria away. Approximately 50 ml of thephage supernatant was then transferred to a fresh tube, and about 12.5ml of PEG solution (20% PEG8000, 3.5 M ammonium acetate) was added andincubated on ice for 2 hours to precipitate phage. The precipitatedphage were centrifuged down and resuspended in 6 ml of the phageresuspension buffer (250 mM NaCl, 100 mM Tris pH8, 1 mM EDTA). Thisphage solution was further purified by centrifuging away the remainingbacteria and precipitating the phage for the second time by adding about1.5 ml of the PEG solution. After a centrifugation step, the phagepellet was resuspended in about 400 μl of PBS. This solution wassubjected to a final centrifugation to rid the solution of any remainingbacterial debris. The resulting phage preparation was titered usingstandard plaque forming assays.

4. Additional Selection and Amplification

In the second round, the amplified phage preparation (about 10¹⁰ pfu)from the first round (section 3C) was used as the input phage to performthe selection and amplification steps (sections 2 and 3). For the Ang-2and Tie-2 elutions, phage from 10 nM and non-specific elutions werecombined and amplified for the third round of selection. The amplifiedphage preparation (about 10⁹ pfu) from the 2^(nd) round in turn was usedas the input phage to perform 3^(rd) round of selection andamplification (sections 2 and 3). After the elution steps (sections 2D,2E, and 2F) of the 3^(rd) round, a small fraction of the eluted phagewas plated out as in the plaque formation assay (section 3C). Individualplaques were picked and placed into 96 well microtiter plates containing100 μl of TE buffer in each well. These master plates were incubated at4° C. overnight to allow phage to elute into the TE buffer.

5. Clonal Analysis

The phage clones were analyzed by phage ELISA and DNA sequencing. Thesequences were ranked based on the combined results from these twoassays.

A. Phage ELISA

An XL-1 Blue MRF′ culture was grown until OD₆₀₀ reached about 0.5. Aboutthirty μl of this culture was aliquoted into each well of a 96-wellmicrotiter plate. About 10 μl of eluted phage (section 4) was added toeach well and allowed to infect bacteria for about 15 minutes at roomtemperature. About 100 μl of LB media containing approximately 12.5μg/ml of tetracycline and approximately 50 μg/ml of ampicillin wereadded to each well. The microtiter plate was then incubated with shakingovernight at about 37° C. The recombinant Ang-2 protein (about 1 μg/mlin PBS) was allowed to bind to the 96 well Maxisorp plates (NUNC)overnight at about 4° C. As a control, the pure streptavidin was coatedonto a separate Maxisorp plate at about 2 μg/ml in PBS.

On the following day, liquid in the protein coated Maxisorp plates wasdiscarded, and each well was blocked with about 300 μl of 5% milksolution at about 4° C. overnight (alternatively, 1 hour at roomtemperature). The milk solution was then discarded, and the wells werewashed three times with the PBST solution. After the last washing step,about 50 μl of PBST-4% milk was added to each well of the protein coatedMaxisorp plates. About 50 μl of overnight cultures from each well in the96 well microtiter plate was transferred to the corresponding wells ofthe Ang-2 coated plates as well as the control streptavidin coatedplates. The 100 μl mixture in the each type of plate was incubated forbout 1 hour at room temperature. The liquid was discarded from theMaxisorp plates, and the wells were washed about three times with PBST.The HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) wasdiluted to about 1:7,500, and about 100 μl of the diluted solution wasadded to each well of the Maxisorp plates for an approximately 1 hourincubation at room temperature. The liquid was again discarded and thewells were washed about five times with PBST. About 100 μl of TMBsubstrate (Sigma) was then added to each well, and the reaction wasstopped with about 50 μl of the 5N H₂SO₄ solution. The OD₄₅₀ was read ona spectrophotometer (Molecular Devices).

B. Sequencing of the Phage Clones

For each phage clone, the sequencing template was prepared using PCR.The following oligonucleotide pair was used to amplify an approximately500 nucleotide fragment:

Primer 1: 5′-CGGCGCAACTATCGGTATCAAGCTG-3′ (SEQ ID NO: 54) Primer 2:5′-CATGTACCGTAACACTGAGTTTCGTC-3′ (SEQ ID NO: 55)

The following mixture was prepared for each clone:

Reagents Volume (μL)/Tube dH₂O 26.25 50% glycerol 10 10X PCR Buffer (w/oMgCl₂) 5 25 mM MgCl₂ 4 10 mM dNTP mix 1 100 μM primer 1 0.25 100 μMprimer 2 0.25 Taq polymerase 0.25 Phage in TE (section 4) 3 Finalreaction volume 50

For PCR, a thermocycler (GeneAmp PCR System 9700, Applied Biosystems)was used to run the following program: 94° C. for 5 minutes; (94° C. for30 sec, 55° C. for 30 sec, 72° C. for 45 sec.)×30 cycles; 72° C. for 7minutes; cool to 4° C. The PCR product from each reaction was purifiedusing the QIAquick Multiwell PCR Purification kit (Qiagen), followingthe manufacturer's protocol. Purified PCR product was then assayed byrunning about 10 μl of each PCR reaction mix with about 1 μl of dye(10×BBXS agarose gel loading dye) on a 1% agarose gel. The remainingproduct was then sequenced using the ABI 377 Sequencer (Perkin Elmer)following the manufacturer recommended protocol.

6. Sequence Ranking and Consensus Sequence Determination

A. Sequence Ranking and Analysis

The peptide sequences that were translated from variable nucleotidesequences (section 5B) were correlated to ELISA data. The clones thatshowed a high OD₄₅₀ in the Ang-2 coated wells and a low OD₄₅₀ in thestreptavidin coated wells were given a higher priority ranking. Thesequences that occurred multiple times were also given a high priorityranking. Candidate sequences were chosen based on these criteria forfurther analysis as peptides or peptibodies.

B. Consensus Sequence Determination

Three-different-classes of consensus motifs were generated from theTN8-IX library as follows:

KRPCEEXWGGCXYX (SEQ ID NQ: 56) KRPCEEXFGGCXYX (SEQ ID NO: 57)XXXCXDXYWYCXXX (SEQ ID NO: 61) XXXCXDXYTYCXXX (SEQ ID NO: 62)XXXCXDXFWYCXXX (SEQ ID NO: 63) XXXCXDXFTYCXXX (SEQ ID NO: 64)XXXCXWDPWTCEXM (SEQ ID NO: 58)

One consensus motif was generated from the TN12-I library:

WSXCAWFXGXXXXXCRRX (SEQ ID NO: 59)

For all consensus motif sequences, the underlined “core amino acidsequences” from each consensus sequence were obtained by determining themost frequently occurring amino acid in each position. “X” refers to anynaturally occurring amino acid. The two cysteines adjacent to the coresequences were fixed amino acids in the TN8-IX and TN12-I libraries.

The peptides identified as binding to Ang-2 are set forth in Table 3below.

TABLE 3 Ang-2 Binding Peptides Peptide Seq Id No. Sequence TN8-8 1KRPCEEMWGGCNYD TN8-14 2 HQICKWDPWTCKHW TN8-Con1 3 KRPCEEIFGGCTYQTN8-Con4 4 QEECEWDPWTCEHM TN12-9 5 FDYCEGVEDPFTFGCDNH L1 6KENPLDELEETLYEQFTFQQ C17 7 QYGCDGFLYGCMIN

EXAMPLE 4 Construction of DNA Encoding Peptibodies

The modified peptides selected as potentially inhibitory to Ang-2:Tie-2binding (see Table 3) were used to construct fusion proteins in whicheither a monomer of each peptide or a tandem dimer of each peptide (witha linker between the monomer units) was fused in-frame to DNA encoding alinker followed by the Fc region of human IgG1. Each modified peptidewas constructed by annealing pairs of oligonucleotides (“oligos”) togenerate a polynucleotide duplex encoding the peptide together with alinker comprised, depending on the peptide, of either five glycineresidues, eight glycine residues or one lysine residue; these constructswere generates as NdeI to XhoI fragments. These duplex polynucleotidemolecules were ligated into the vector (pAMG21-Fc N-terminal, describedfurther below) containing the human Fc gene, which had been previouslydigested with NdeI and XhoI. The resulting ligation mixtures weretransformed by electroporation into E. coli strain 2596 cells (GM221,described further below) using standard procedures. Clones were screenedfor the ability to produce the recombinant protein product and topossess the gene fusion having a correct nucleotide sequence. A singlesuch clone was selected for each of the modified peptides (i.e.,Fc-peptide fusion products).

Construction of pAMG21-Fc N-Terminal Vector

pAMG21

Expression plasmid pAMG21 (ATCC No. 98113) is derived from expressionvector pCFM1656 (ATCC No. 69576) and the expression vector systemdescribed in U.S. Pat. No. 4,710,473, by following the proceduredescribed in published International Patent Application WO 00/24782 (seethe portion of Example 2 therein extending from pages 100-103, as wellas FIGS. 17A and 17B).

Fc N-terminal Vector

The Fc N-terminal vector was created using E. coli strain 3788, pAMG21Tpo_Gly5_Fc monomer, as a template. Information on the cloning of thisstrain can be found in WO 00/24782 (See Example 2 and FIG. 10 therein).A 5′ PCR primer (described further below) was designed to remove the Tpopeptide sequence in pAMG Tpo Gly5 and replace it with a polylinkercontaining ApaLI and XhoI sites. Using strain 3788 as a template, PCRwas performed with Expand Long Polymerase, using the oligonucleotide ofSEQ ID NO: 8, below, as the 5′ primer and a universal 3′ primer, SEQ IDNO: 9, below. The resulting PCR product was gel purified and digestedwith restriction enzymes NdeI and BsrGI. Both the plasmid and thepolynucleotide encoding the peptide of interest together with its linkerwere gel purified using Qiagen (Chatsworth, Calif.) gel purificationspin columns. The plasmid and insert were then ligated using standardligation procedures, and the resulting ligation mixture was transformedinto E. coli cells (strain 2596). Single clones were selected and DNAsequencing was performed. A correct clone was identified and this wasused as a vector source for the modified peptides described herein.

5′ Primer: ACAAACAAACATATGGGTGCACAGAAAGCGOCCGCA (SEQ ID NO: 8)AAAAAACTCGAGGGTGGAGGCGGTGGGGACA 3′ Primer: GGTCATTACTGGACCGGATC (SEQ IDNO: 9)

In addition to making these modified peptides as N-terminal fusions toFc (N-terminal peptibodies), some of them were also made as C-terminalfusion products (C-terminal peptibodies). The vector used for making theC-terminal fusions is described below.

Construction of Fc C-terminal Vector

The Fc C-terminal vector for modified peptides was created using E. colistrain 3728, pAMG21 Fc_Gly5_Tpo monomer, as a template. Information onthe cloning of this strain can be found in WO 00/24782 (See Example 2and FIG. 7 therein). A 3′ PCR primer (SEQ ID NO: 10) was designed toremove the Tpo peptide sequence and to replace it with a polylinkercontaining ApaLI and XhoI sites. Using strain 3728 as a template, PCRwas performed with Expand Long Polymerase using a universal 5′ primer(SEQ ID NO: 11) and the aforementioned 3′ primer. The resulting PCRproduct was gel purified and digested with restriction enzymes BsrGI andBamH. Both the plasmid and the polynucleotide encoding each peptides ofinterest with its linker were gel purified via Qiagen gel purificationspin columns. The plasmid and insert were then ligated using standardligation procedures, and the resulting ligation mixture was transformedinto E. coli (strain 2596) cells. Single clones were selected and DNAsequencing was performed. A correct clone was identified and used as asource of vector for modified peptides described herein.

5′ Primer: CGTACAGGTTTACGCAAGAAAATGG (SEQ ID NO: 10) 3′ Primer:TTTGTTGGATCCATTACTCGAGTTTTTTTGCGGC (SEQ ID NO: 11)CGCTTTCTGTGCACCACCACCTCCACCTTTAC

GM221 (#2596). Host strain #2596, used for expressing Fc-peptide fusionproteins, is an E. coli K-12 strain modified to contain the luxpromoter, and both the temperature sensitive lambda repressor cI857s7 inthe early ebg region and the lacI^(Q) repressor in the late ebg region.The presence of these two repressor genes allows the use of this hostwith a variety of expression systems The ATCC designation for thisstrain is 202174.

EXAMPLE 5 Production of Peptibodies

Expression in E. coli. Cultures of each of the pAMG21-Fc fusionconstructs in E. coli GM221 were grown at 37° C. in Terrific Brothmedium (See Tartof and Hobbs, “Improved media for growing plasmid andcosmid clones”, Bethesda Research Labs Focus, Volume 9, page 12, 1987,cited in aforementioned Sambrook et aL reference). Induction of geneproduct expression from the luxPR promoter was achieved following theaddition of the synthetic autoinducer, N-(3-oxohexanoyl)-DL-homoserinelactone, to the culture medium to a final concentration of 20 nanogramsper milliliter (ng/ml). Cultures were incubated at 37° C. for anadditional six hours. The bacterial cultures were then examined bymicroscopy for the presence of inclusion bodies and collected bycentrifugation. Refractile inclusion bodies were observed in inducedcultures, indicating that the Fc-fusions were most likely produced inthe insoluble fraction in E. coli. Cell pellets were lysed directly byresuspension in Laemmli sample buffer containing 10% β-mercaptoethanoland then analyzed by SDS-PAGE. In most cases, an intensecoomassie-stained band of the appropriate molecular weight was observedon an SDS-PAGE gel.

Purification. Cells were broken in water (1/10) using high pressurehomogenization (two passes at 14,000 PSI), and inclusion bodies wereharvested by centrifugation (4000 RPM in a J-6B centrifuge, for onehour). Inclusion bodies were solubilized in 6 M guanidine, 50 mM Tris,10 mM DTT, pH 8.5, for one hour at a 1/10 ratio. For linear peptidesfused to Fc, the solubilized mixture was diluted twenty-five times into2 M urea, 50 mM Tris, 160 mM arginine, 2 mM cysteine, pH 8.5. Theoxidation was allowed to proceed for two days at 4° C., allowingformation of the disulfide-linked compound (i.e., Fc-peptide homdimer).For cyclic peptides fused to Fc, this same protocol was followed withthe addition of the following three folding conditions: (1) 2 M urea, 50mM Tris, 160 mM arginine, 4mM cysteine, 1 mM cystamine, pH 8.5; (2) 4 Murea; 20% glycerol, 50 mM Tris, 160 mM arginine, 2.mM cysteine, pH 8.5;and (3) 4 M urea, 20% glycerol, 50 mM Tris, 160 mM arginine, 4 mMcysteine, imM cystamine, pH 8.5. The refolded protein was dialyzedagainst 1.5 M urea, 50 mM NaCl, 50 mM Tris, pH 9.0. The pH of thismixture was lowered to pH 5 with acetic acid. The precipitate wasremoved by centrifugation, and the supernatant was adjusted to a pH offrom 5 to 6.5, depending on the isoelectric point of each fusionproduct. The protein was filtered and loaded at 4° C. onto anSP-Sepharose HP column equilibrated in 20 mM NaAc, 50 mM NaCl at the pHdetermined for each construct. The protein was eluted using a 20-columnvolume linear gradient in the same buffer ranging from 50 mM NaCl to 500mM NaCl. The peak was pooled and filtered.

The peptibodies generated using the procedures above are set forth inTable 4 below.

TABLE 4 Peptibody Peptibody Sequence L1 (N)GAQKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 12) L1 (N) WTMKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 13) L1 (N) 1K WTMKFNPLDELEETLYEQFTFQQGSGSATGGSGSTASS GSGSATHLEGGGGG-Fc (SEQ ID NO: 14)2xL1 (N) MGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNP LDELETLYEQFTFQQLEGGGGG-Fc(SEQ ID NO: 15) 2xL1 (N) WT MKFNPLDELEETLYEQFTFQQGGGGGGGKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 16) Con4 (N)MGAQQEECEWDPWTCEHMLEGGGGG-Fc (SEQ ID NO: 17) Con4 (N) 1K-WTMQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSAT HLEGGGGG-Fc (SEQ ID NO: 18) 2xCon4(N) 1K MGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSG SATHQEECEWDPWTCEHMLEGQQGG-Fc(SEQ ID NO: 19) L1 (C) M-Fc-GGGGGAQKFNPLDELEETLYEQFTFQQLE (SEQ ID NO:20) L1 (C) 1K M-Fc-GGGGGAQGSGSATGQSGSTASSGSGSATHKF NPLDELEETLYEQFTFQQLE(SEQ ID NO: 21) 2xL1 (C) M-Fc-GGGGGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPLDELEETLYEQFTFQQLE (SEQ ID NO: 22) Con4 (C)M-Fc-GGGGGAQQEECEWDPWTCEHMLE (SEQ ID NO: 23) Con4 (C) 1KM-Fc-GGGGGAQGSGSATGGSGSTASSGSGSATHQE ECEWDPWTCEHMLE (SEQ ID NO: 24)2xCon4 (C) 1K M-Fc-GGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 25) Con4-L 1 (N)MGAQEECEWDPWTCEHMGGGGGGGGKFNPLDELEETLYQFTFQQGSGSATGGSGSTASSGSGSATHLEGGGG G-Fc (SEQ ID NO: 26) Con4-L1 (C)M-Fc-GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELEETYEQFTFQQGGGGQQEECEWDPWTCEHM LE (SEQ ID NO: 27) TN-12-9 (N)MGAQ-FDYCEGVEDPFTFGCDNHLE-GGGGG-Fc (SEQ ID NO: 28) C17 (N)MGAQ-QYGCDGFLYGCMINLE-GGGGG-Fc (SEQ ID NO: 29) TN8-8 (N)MGAQ-KRPCEEMWGGCNYDLEGGGG-Fc (SEQ ID NO: 30) TN8-14 (N)MGAQ-HQICKWDPWTCKHWLEGGG-Fc (SEQ ID NO: 31) Con1 (N)MGAQ-KRPCEEIFGGCTYQLEGGGGG-Fc (SEQ ID NO: 32)

In Table 4, “Fc” refers to the human Fc IgG1 sequence. Column two setsforth the amino acid sequence of the peptibody. The Fc portion thereofis labeled “Fc”, and is as set forth in SEQ ID NO: 60 below. It will beappreciated that where a label is used, for example, “Con4” or “Con-4”,this refers to the Con-4 peptide, whereas use of the suffix “C”, “(C)”,or “—C”; or “N”, “(N)”, or “—N” thereon indicates that the molecule is apeptibody as described herein. The suffixes “N”, “(N)”, or “—N” in apeptibody name indicate that the Ang-2-binding peptide (or peptides)is/are N-terminal to the Fc domain, and the suffixes “C”, “(C)” or “—C”indicate that the Ang-2-binding peptide (or peptides) is/are C-terminalto the Fc domain. Furthermore, 2xCon4 (C) 1K, as defined in SEQ ID NO:25, may also be referred to without the “1K” suffix herein.

The amino acid sequence of the Fc portion of each peptibody is asfollows (from amino terminus to carboxyl terminus):

(SEQ ID NO: 60) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The DNA sequence (SEQ ID NOs: 33-53) encoding peptibodies correspondingto peptibody SEQ ID NOs: 12-32, respectively, in Table 4) is set forthbelow:

SEQ ID NO: 33 ATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAATGGATCC SEQ ID NO: 34ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGCTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATTCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCGTGCCCCCATCCCGGOATGAGGTGACCAAGAACCAGGTCAGCCTGACCTGCGTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGOAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGOCTCCTTCTTCCTGTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAATAASEQ ID NO: 35 ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA SEQ ID NO: 36ATGGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGTGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCCACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCAACTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCTGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGTAAATAA SEQ ID NO:37 ATGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCCACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCAACTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA SEQ ID NO: 38ATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCATACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATAA SEQ IDNO: 39 ATGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAATAA SEQID NO:40 ATGGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCAGGAAGAATGCGAATGGGACCCARTGGACTTGCGAACACATGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGCTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCGAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAATAASEQ ID NO: 41 ATGGACAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGTGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCCGCTGGACGAGCTGGAAGAGACTCTGTACGAACAGTTTACTTTTCAACAGCTCGAGTAA SEQ ID NO:42ATGGACAAAACTGACACATGTCCACCTTGCCCAGCACCTGAACTCGTGGGGGGACCGTGAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAGGAGTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATGTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCGGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCGTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTGTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTCTGTTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTTCACTlTTCCAGCAACTCGAGTAA SEQ ID NO: 43ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGTGGTGGTGGTGGTGGCGGTGGTAAGTTCAACCCACTGGATGAGCTGGAAGAGACTCTGTATGAACAGTTCACTTTCCAGCAAC TCGAGTAA SEQ ID NO:44 ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTC GAGTAA SEQ ID NO: 45ATGGACAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA SEQ ID NO: 46ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGTAGGTGGTGGTGCACAGCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGCGCGACTCATCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAA CACATGCTCGAGTAA SEQID NO: 47 ATGGGTGCACAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGGGTGGTGGTGGTGGTGGCGGTGGTAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATCTCGAGCGTGGAGGCGGTGGgGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCTCCCATCCCGGGATGAGCTGACCAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTA AATAA SEQ ID NO: 48ATGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGTGGAGGTGGTGGTGCACAGGGATCCGGTTCTGCTACTGGTGGTTCCGGCTCCACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATAAATTCAACCCGCTGGACGAACTGGAAGAAACTCTGTACGAACAGTTCACTTTCCAGCAGGGTGGTGGCGGTGGTCAGGAAGAATGCGAATGGGACCCATGGACTTGCGAACACATGCTCGAGTAA SEQ ID NO: 49:ATGGGTGCACAGTTCGACTACTGCGAAGGTGTTGAAGACCCGTTCACTTTCGGTTGCGACAACCACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCADCTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTAAATAASEQ ID NO: 50 ATGGGTGCACAGCAGTACGGTTGCGACGGTTTTCTGTACGGTTGCATGATCAACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCAGAACCACGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGVACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATAA SEQ ID NO: 51ATGGGTGCACAGAAACGCCCATGCGAAGAAATGTGGGGTGGTTGCAACTACGACCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATAA SEQ ID NO:52 ATGGGTGCACAGCACCAGATCTGCAAATGGGACCCGTGGACCTGCAAACACTGGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCTGTCTGGACTCCGACGGCTCCTTCTTCCGTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATAA SEQ ID NO: 53ATGGGTGCACAGAAACGTCCARTGCGAAGAAATCTTCGGTGGTTGCACCTACCAGCTCGAGGGTGGAGGCGGTGGGGACAAAACTCACACATGTCCACCTTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGAGCCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATAA

EXAMPLE 6 Peptibody Assays

Fourteen of the peptibodies were tested using the neutralization ELISA,and three of the peptibodies were tested using the affinity ELISA. Theresults are set forth in Table 5.

TABLE 5 hAng-2 mAng-2 IC 50 EC 50 EC 50 hAng-1 Peptibody (nM) (nM) IC 50(nM) (nM) IC 50 (nM) EC 50 (nM) 2xCon4 (C) 1K 0.04 0.02 Con4-L1 (C) 0.050.04 Con4 (C) 0.20 0.30 2xL1 (N) 0.65 0.80 Con4 (N) 0.85 0.03 0.72 0.07No Inhibition No Binding 2xL1 (C) 0.90 1.0 Con4 (N) 1K- 1.9 WT L1 (N) 611 No Inhibition C17 (N) 9 13 No Inhibition 12-9 (N) 21 7.7 NoInhibition Con1 (N) 26 ~200 No Inhibition 8-14 (N) 45 33 No InhibitionL1 (C) 65 37 8-8 (N) 80 ~700 No Inhibition Negative No No No InhibitionNo No Inhibition No Binding Control Inhibition Binding Binding Peptibody4883

The amino acid sequence of negative control peptibody 4883 is as follows(the Fc portion is underlined, the linker is “GGGGG”, and the peptideportion is in bold):

(SEQ ID NO: 243) MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-GGGGG-CTAGYHWNSDCECCRRN

It will be appreciated that use of the term “No Inhibition” herein isnot meant to indicate that the compounds have no inhibitory qualities.Rather, “No Inhibition” as used herein refers to those compounds whichwhen tested using the neutralization ELISA assay under the conditionsdescribed herein exhibited an IC₅₀ value of greater than 1000 nM, whichwas the highest concentration at which these compounds were screened.While significant inhibitory qualities were not observed for themolecules labeled as exhibiting “no inhibition”, it will be appreciatedthat those molecules may in fact demonstrate inhibitory qualities underdifferent assay conditions, or in different assays. In a preferredembodiment, it will be appreciated that the invention relates topeptibodies that have inhibitory qualities using the assays describedherein.

Two of the peptibodies were tested using the affinity BIAcore assay (asdescribed in Example 2). The results are set forth in Table 6 below.

TABLE 6 Peptibody (Pb) Affinities for hAng-2 and mAng-2 hAng-2 mAng-2K_(D) K_(D) Peptibody (nM) k_(a) (1/Ms) k_(d) (1/s) (nM) k_(a) (1/Ms)k_(d) (1/s) Pb L1 (N) 3.1 2.9 × 10⁵ 9.1 × 10⁻⁴ 0.42 5.6 × 10⁵ 2.3 × 10⁻⁴Con4 (N) 0.67 3.3 × 10⁵ 2.2 × 10⁻⁴ 0.60 7.3 × 10⁵ 4.4 × 10⁻⁴ TN12-9 8.21.2 × 10⁵ 1.0 × 10⁻³ 0.32 7.2 × 10⁵ 2.3 × 10⁻⁴ (N)

EXAMPLE 7 Therapeutic Efficacy Studies With Systemically AdministeredAng-2 Peptibody

Ang-2 peptibody, TN8-Con4-C, was administered subcutaneously to A431tumor-bearing mice at a once-per-day schedule 72 hours after tumorchallenge. The doses of peptibody used were 1000, 200, 40 and8ug/mouse/day. A total of 20 doses was given to all animals. Tumorvolumes and body weights were recorded three times/week. At the end ofthe study, animals were sacrificed, and their sera were collected formeasuring peptibody levels by ELISA. Tumors and a panel of normaltissues were collected from all groups.

The results are shown in FIG. 1. As can be seen, significant differencesin tumor growth were observed between the Ang-2 peptibody treated groupand vehicle control. All four doses of Ang-2 peptibody inhibited tumorgrowth as compared to vehicle controls (p<0.0001 vs. vehicle controlusing repeated measure ANOVA). In contrast, tumors in the control groupcontinued to grow at a much greater rate. Treatment with this peptibodyhad no significant effect on terminal body weights, organ weights orhematology parameters of the animals treated at the above doses.

EXAMPLE 8

1. Construction of Ang-2 Secondary Peptide Libraries

A. Electrocompetent E.coli Cells

Epicurian Coli® XL1-Blue MRF′ electroporation competent cells(Stratagene #200158) were purchased from Stratagene (Stratagene CloningSystems, La Jolla, Calif.).

B. Modification of pCES1 Vector

PCR was performed using Extend Long Template PCR Systems (RocheDiagnostics Corp., Indianapolis, Ind.) with 1 μg of pCES1 vector(TargetQuest Inc.) as a template. PCR mixture volume was 100 μl whichcontained 1×PCR buffer, 200 nM of each of the two primers:5′-CAAACGAATGGATCCTCATTAAAGCCAGA-3′ (SEQ ID NO: 244) and5′-GGTGGTGCGGCCGCACTCGAGACTGTTGAAAGTTGTTTAGCA-3′ (SEQ ID NO: 245), 200nM DNTP, and 3 units (U) of Tag DNA polymerase. The TRIO-Thermoblock(Biometra) PCR system was run as follows: 94° C. for 5 minutes; 30cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 45seconds; and 72° C. for 10 minutes; cool to 4° C.

The PCR products were then run on a 1% agarose gel and purified withQIAGEN Spin Column (QIAGEN Inc., Valencia, Calif.) according to themanufacturer's protocols. A second PCR reaction was performed with 5 μlof PCR products and 200 nM of each of the two primer5′-CAAACGAATGGATCCTCATTAAAGCCAGA-3′ (SEQ ID NO: 246) and5′-AACACAAAAGTGCACAGGGTGGAGGTGGTGGTGCGGCCGCACT-3′ (SEQ ID NO: 247) underthe same PCR conditions as described above.

The PCR products and original pCES1 vector were then digested separatelyin a 100 μl reaction containing 1× NEB2 buffer, 60 U of ApaLI (NewEngland Biolabs, Beverly, Mass.), 60 U of BamHI (New England Biolabs) at37° C. for 1 hour. The digested DNA was then purified using a QIAGENSpin Column and ligated together in a 40 μl reaction containing 1×ligation buffer and 40 U of T4 DNA ligase (New England Biolabs) at roomtemperature overnight.

The vectors were transfected into E. coli and incubated at 37° C.overnight. Isolated single colonies were selected and plasmid was thenpurified using a QIAGEN Spin Column. The correct insert was confirmed byDNA sequencing.

C. Preparation of Vector DNA

One microgram of modified pCES1 vector DNA (from section 1B above) wastransformed into 40 μl of electrocompetent XL1-blue E.coli (from section1A above) using the Gene Pulser II (BIO-RAD, Hercules, Calif.) set at2500V, 25 μF, and 200 ohms. The transformed bacteria sample was thentransferred immediately into a tube containing 960 μl of SOC (2%tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 20 mM glucose, 10mM MgSO₄, 10 mM MgCl₂), and the culture was allowed to grow at 37° C.with shaking for 1 hour.

The cells were then spread onto the 2×YTAGT (2×YT with 100 ug/mlampicillin, 12.5 ug/ml tetracycline and 2% glucose) agar plate andincubated at 37° C. overnight. A single colony was confirmed bysequencing and used to inoculate 2 liters of 2×YTAGT media at 37° C.with shaking overnight. The plasmid vector DNA was purified with QIAGENPlasmid Maxi Kit according to the manufacturer's protocols.

D. Digestion of Vector DNA

Total about 2000 micrograms of vector DNA (from section IC above) wasdigested in 5000 μl reaction containing 1× NEB buffer2, 300 U of ApaLI,and 300 U of XhoI at 37° C. overnight. The restriction digest reactionwas incubated overnight at 37° C. and analyzed in a pre-made 0.8%agarose gel (Embi Tec, San Diego, Calif.). The linearized vector DNA wasthen excised from the gel and extracted with QIAquick Gel Extraction Kit(QIAGEN Inc.) according to the manufacturer's directions.

E. Preparation of Library Oligonucleotides

Six library oligonucleotides (1 fixed and 5 doped) were designed basedon the sequences that derived from the results described above. The onefixed library oligonucleotides was:

(SEQ ID NO: 248) 5′-CACAGTGCACAGGGTNNKNNKNNKNNKNNKNNKNNKSARTGGGATCCGTGGASCNNKNNKNNKNNKNNKNNKNNKCATTCTCTCGAGATCA-3′ (library number 20);and two of the 70% doped library oligonucleotides were as follows:

(SEQ ID NO: 249) 5′-CACAGTGCACAGGGTNNKNNKNNKaaKcgKccKNNKgaKgaKatKttKggKggKNNKacKtaKcaKNNKNNKNNKCATTCTCTCGAGATCA-3′ (library number 27);(SEQ ID NO: 250) 5′-CACAGTGCACAGGGTNNKaaKttKaaKccKctKgaKgaKctKgaKgaKacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATC A-3′ (library number99);

Lower case letters represent a mixture of 70% of the indicated base and10% of each of the other three nucleotides). The other three of the 91%doped library oligonucleotides were as follows:

(SEQ ID NO: 251) 5′-CACAGTGCACAGGGTNNKNNKNNKcaKgaKgaKTGCgaKtgKgaKccKtgKacKTGCgaKcaKatKNNKNNKNNKCATTCTCTCGAGATCA-3′ (library number 94);(SEQ ID NO: 252) 5′-CACAGTGCACAGGGTNNKttKgaKtaKNNKgaKggKgtKgaKgaKccKttKacKttKggKNNKgaKaaKcaKNNKCATTCTCTCGAGATCA-3′ (library number 25); and(SEQ ID NO: 253) 5′-CACAGTGCACAGGGTNNKaaKttKaaKccKctKgaKgaKctKgaKgaKacKctKtaKgaKcaKttKacKttKcaKcaKNNKCATTCTCTCGAGATC A-3′ (library number26);

For the oligos above, those skilled in the art will appreciate that “N”indicates that each of the four nucleotides (A, T, C, and G) are equallyrepresented during oligo synthesis, and “K” indicates that nucleotides Gand T were equally represented during oligo synthesis. Lower caseletters represent a mixture of 91% of the indicated base and 3% of eachof the other three nucleotides. Each of these oligonucleotides was usedas templates in PCR.

Expand High Fidelity PCR System kit (Roche Diagnostics Corp.) was usedfor the PCR reactions. Each library oligo was amplified in a ninety sixwell 50 μl PCR reaction which contained 1 nM of a libraryoligonucleotide, 1×PCR buffer, 300 nM of each of the primers:

5′-CACAGTGCACAGGGT-3′; (SEQ ID NO: 254) and 5′-TGATCTCGAGAGAATG-3′,;(SEQ ID NO: 255)

200 μM dNTP, 1.5 mM MgCl₂, and 350 U of the Expand polymerase. Thethermocycler (GeneAmp PCR System 9700, Applied Biosystems) was used torun the following program: 94° C. for 5 minutes; 25 cycles of (94° C.for 30 seconds, 52.5° C. for 60 seconds, 72° C. for 30 seconds); 72° C.for 10 minutes; cool to 4° C. The free nucleotides were then removedusing the QIAquick PCR Purification Kit (QIAGEN Inc. Cat#28104)according to the manufacturer's protocols.

F. Digestion of Library Oligonucleotides

For each library the PCR products (section IE) were digested in a 1200μl reaction that contained 1× NEB buffer2, 750 U of ApaLI, and 750 U ofXhoI at 37° C. overnight. The digested DNA was separated on a pre-made3% agarose gel (Embi Tec). The DNA band of interest from each reactionwas cut from the gel and extracted with COSTAR Spin-X centrifuge tubefilter, 0.22 μm cellulose acetate (Corning Inc., Cat#8160).

G. Ligation of Vector with Library Oligonucleotides

The 450 μl ligation reaction contained the linearized vector (section1D) and each digested library PCR product (section 1F) at 1:5 molarratio, 1× NEB ligation buffer, and 20,000 U of the T4 DNA ligase at 16°C. overnight. The ligated products were incubated at 65° C. for 20minutes to inactivate the T4 DNA ligase and further incubated with 100 UNotI at 37° C. for 2 hours to minimize vector self-ligation. The ligatedproducts were then purified by a standard phenol/chloroform extraction(Molecular Cloning: A Laboratory Manual, Maniatis et al., 3^(rd)Edition, Cold Spring Harbor Laboratory Press, 2000) and resuspended in120 μl of H₂O.

H. Electroporation Transformation

For each library, twelve electroporation reactions were performed. Foreach transformation, 10 μl of the ligated vector DNA (section 1G) and300 μl of XL1-BLUE MRF′ cells (section 1A) were mixed in a 0.2-cmcuvette (BIO-RAD). The resulting mixture was pulsed by the Gene Pulser Hsetting at 2500 V, 25 uF, and 200 ohms. The transformed bacteria fromthe twelve electroporation reactions were then combined and transferredinto a flask containing 26 ml of SOC for incubation at 37° C. for 1hour. The cells were added to 450 ml 2×YTAG and grown at 37° C. withshaking for 5 hours. The cells were centrifuged at 4000 rpm for 15minutes at 4° C. The cell pellets were then resuspended in 12 ml of 15%glycerol/2×YT and stored at −80° C. This was the primary stock of thelibraries. Titers showed library sizes of 5.0×10⁹ (library number 20),3.3×10¹⁰ (library number 94), 4.7×10⁹ (library number 25), 5.0×10⁹(library number 26), 3.0×10⁹ (library number 27), and 4.2×10⁹ (librarynumber 99) independent transformants.

2. Amplification of the Libraries

A. Making Secondary Stock of the Libraries

From the primary library cell stock (from section 1H above), sufficientcells to cover a 10× excess of each library size were used to inoculate2×YTAGT (2YT with 100 ug/ml ampicillin, 12.5 ug/ml tetracycline and 2%glucose) media so that the starting OD₆₀₀ was 0.1. The cultures wereallowed to grow at 37° C. with shaking for several hours until theOD₆₀₀=0.5. A one-tenth aliquot from each library was taken out and grownup in separate flasks for another two hours at 37° C. These sub-cultureswere then centrifuged at 4000 rpm using a Beckman JA-14 rotor for 10minutes at 4° C., and the bacteria pellets resuspended in 7.0 ml (foreach library) of 15% glycerol/2×YT for storage at −80° C.

B. Phage Induction

M13KO7 helper phage aliquots (Amersharn Pharmacia Biotech) were added tothe remaining bacteria cultures at OD₆₀₀=0.5 (from Section 2A above) tothe final concentration of 3×10⁹ pfu/ml. The helper phage were allowedto infect bacteria at 37° C. for 30 minutes without shaking and 30minutes with slow shaking. The infected cells were centrifuged with 5000rpm for 15 minutes at 4° C. The cell pellets were resuspended in thesame volume (from section 2A above) with the 2×YTAK media (2YT with 100ug/ml ampicilliri and 40 ug/ml kanamycin). The phagemid production wasallowed to occur at 30° C. overnight while shaking.

C. Harvest of Phage

The bacteria cultures from section 2B above were centrifuged at 5000 rpmfor 15 minutes at 4° C. The supernatants were then transferred into newbottles, and 0.2 volume of 20% PEG/2.5 M NaCl were added and incubatedon ice for 1 hour to precipitate the phagemids. Precipitated phagemidswere centrifuged at 10,000 rpm for 30 minutes at 4° C. and carefullyresuspended with 100 ml of cold PBS. The phagemid solution was furtherpurified by centrifuging away the remaining cells with 4000 rpm for 10minutes at 4° C. and precipitating the phagemids by adding 0.2 volume of20% PEG/2.5 M NaCl. The phagemids were centrifuged at 10,000 rpm for 30minutes at 4° C., and the phagemid pellets resuspended with 18 ml ofcold PBS. Six ml of 60% glycerol solution was added to the phagemidsolution for storage at −80° C. The phagemid titers were determined by astandard procedure (Molecular Cloning, Maniatis et al 3^(rd) Edition).

3. Selection of Ang-2 Binding Phage

A. Immobilization of Ang-2 on Magnetic Beads

The biotinylated Ang-2 (from section 3A above) was immobilized on theDynabead M-280 Streptavidin (DYNAL, Lake Success, N.Y.) at aconcentration of 2000 ng Ang-2 protein per 100 μl of the bead stock fromthe manufacturer. After drawing the beads to one side of a tube using amagnet and pipetting away the liquid, the beads were washed twice withphosphate buffer saline (PBS) and resuspended in PBS. The biotinylatedAng-2 protein was added to the washed beads at the above concentrationand incubated with rotation for 1 hour at room temperature. The Ang-2coated beads were then blocked by adding BSA to 2% final concentrationand incubating overnight at 4° C. with rotation. The resulting Ang-2coated beads were then washed twice with PBST (PBS with 0.05% Tween-20)before being subjected to the selection procedures.

B. Selection Using the Ang-2 Coated Beads

About 1000-fold library equivalent phagemids (from section 2C above)were blocked for one hour with 1 ml of PBS containing 2% BSA. Theblocked phagemid sample was subjected to three negative selection stepsby adding it to blank beads (same beads as section 3A but with no Ang-2protein coating), and this mixture was incubated at room temperature for15 minutes with rotation. The phagemid containing supernatant was drawnout using magnet and transferred to a second tube containing blank beads(the same beads as described in section 3A above but without Ang-2protein coated thereon), and this mixture incubated at room temperaturefor 15 minutes with rotation.

The procedure was repeated. The phagemid containing supernatant was thendrawn out using magnet and transferred to a new tube containing Ang-2protein coated beads (from section 3A), and the mixture was incubated atroom temperature for 1 hour with rotation. After the supernatant wasdiscarded, the phagemid-bound-beads were washed 10 times with 2%milk-PBS; 10 times with 2% BSA-PBS; 10 times with PBST and twice withPBS. The phagemids were then allowed to elute in 1 ml of 100 mMtriethylamine solution (Sigma, St. Louis, Mo.) for 10 minutes on arotator. The pH of the phagemid containing solution was neutralized byadding 0.5 ml of 1 M Tris-HCl (pH 7.5). The resulting phagemids wereused to infect 10 ml of freshly grown XL1-Blue MRF′ bacteria (OD600about 0.5) at 37° C. for 30 minutes without shaking and 30 minutes withslow shaking. All of the infected XL1-BLUE MRF′ cells were then platedon a 15×5 cm 2×YTAG plate and incubated at 30° C. overnight.

C. Induction and Harvesting of Phage

A 10 ml aliquot of 2×YTAGT media was added to the plate (from section3B) to resuspend XL1-BLUE MRF′ cells. All XL1-BLUE MRF′ cells werecollected in a tube, and a 250 μl aliquot of these cells was added to 25ml of 2×YTAGT and grown at 37° C. until OD₆₀₀=0.5. The M13KO7 helperphage were added to a final concentration of 3×10⁹ cfu/ml and incubatedat 37° C. for 30 minutes without shaking and 30 minutes with slowshaking. The cells were centrifuged with 5000 rpm for 10 minute at 4° C.and resuspended with 25 ml of 2×YTAK. These bacteria were allowed togrow at 30° C. overnight with shaking. The induced phagemids wereharvest and purified as in section 2C.

D. Second Round Selection

The second round selection was performed as outlined in section 3B to 3Cexcept for the following. About 100-fold library equivalent phagemidsresulting from section 3C was used as the input phagemid. The amount ofbiotinylated Ang-2 protein (section 3A) coat onto the Dynabead M-280Streptavidin was decreased to 20 ng. The phage-bound-beads were thenwashed 10 times with 2% milk-PBS; 10 times with 2% BSA-PBS; 10 timeswith PBST, where the final wash involved 60 minutes incubation at roomtemperature in PBST. The beads were washed twice with PBS. The elutionconditions were same as the first round (section 3B).

E. Third Round Selection

The third round selection was performed as outlined in section 3B to 3Cabove except the following. About 10 fold library equivalent phagemidsresulting from section 3D was used as the input phagemid. About 2ng ofbiotinylated Ang-2 protein (from section 3A) was used to coat onto theDynabead M-280 Streptavidin. The phage-bound-beads were washed 10 timeswith 2% milk-PBS; 10 times with 2% BSA-PBS; 10 times with PBST, wherethe final wash involved 60 minutes incubation at room temperature inPBST. The beads.were washed twice with PBS. The elution conditions weresame as the first round (section 3B).

F. Fourth Round Selection

The fourth round selection was performed as outlined in section 3B to 3Cabove except for the following. Library equivalent phagemids resultingfrom section 3E were used as the input phagemid. The amount ofbiotinylated Ang-2 protein (section 3A) coat onto the Dynabead M-280Streptavidin was decreased to 0.4 ng for libraries 25, 26, and 27. Forlibraries 20 and 94, the coating amount was kept as the third round at 2ng. The library 99 was not carried to the fourth round selection step.The elution conditions were same as the first round (section 3B).

4. Clonal Analysis

A. Preparation of Master Plate

Single colonies from the second round selection were picked andinoculated into 96 well plates containing 120 μl of 2×YTAGT per well.The 96 well plates were incubated in 30° C. shaker for overnight. Fortymicroliters of 60% glycerol were added per well for storage at −80° C.

B. Phagemid ELISA

About 2 μl aliquots of cells from the master plate (from section 4Aabove) were inoculated into a fresh Costar® 96 well plate (Comingincorporated, Corning, N.Y., cat. #9794) which contained 100 μl of2×YTAGT per well, and this new plate of cells was grown at 37° C. untilapproximate OD₆₀₀=0.5.

Forty μl of 2×YTAGT containing M13KO7 helper phage (1.5×10¹³ cfu/ml) wasadded to each well, and the 96 well plate was incubated at 37° C. for 30minutes without shaking and another 30 minutes with slow shaking. Theplate was centrifuged at 2000 rpm (Beckman CS-6R tabletop centrifuge)for 10 minutes at 4° C. The supernatants were removed from the wells,and each cell pellet was resuspended using 150 μl of 2×YTAK per well.The plate was incubated at 30° C. overnight for phagemid expression.

Human Ang-2 protein was coated onto the 96 well Maxisorp plate (NUNC) at1 μg/ml in 1× PBS at 4° C. overnight. As a control, 2% BSA (Sigma) wascoated onto a separate Maxisorp plate. On the following day, theovernight cell cultures were centrifuged at 2000 rpm for 10 minutes at4° C. Ten μl of supernatant from each well was transferred to a new 96well plate which containing BSA/PBS solution to dilute the supernatantat 1:10. The resulting mixtures were incubated for 1 hour at roomtemperature with shaking to block the phagemids. Meanwhile, the Ang-2protein coated plate was blocked with 400 μl of 2% BSA/PBS solution perwell for 1 hour at room temperature while shaking. The BSA solution wasdiscarded, and each well was washed three times with PBS solution. Afterthe last washing step, 100 μl of blocked phagemid solutions was added toeach well of the Ang-2 protein coated plate as well as the control plateand incubated for 1 hour at room temperature with shaking. The liquidwas discarded, and each well was washed three times with PBST solution.One hundred μl of the HRP-conjugated anti-M13 mAb (Amersham PharmaciaBiotech) at 15,000 dilution was added to each well of the Ang-2 proteincoated and control plates, and these plates were incubated for 1 hour atroom temperature with shaking. The liquid was discarded again, and eachwell was washed three times with PBST solution. One hundred μl ofLumiGLO chemiluminescent substrates (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) was added to the wells, and each well was read byLuminoskan Ascent DLRearly machine (Labsystems, Franklin, Mass.).

C. Seguencing of the Phage Clones

PCR reaction was performed using 1 μl of bacteria from each well of themaster plate (section 4A) as a template. The volume of each PCR mixturewas 50 μl which contains 1× PCR buffer, 300 nM of each of the twoprimers:

5′-GTTAGCTCACTCATTAGGCAC-3′ (SEQ ID NO: 256) and5′-GTACCGTAACACTGAGTTTCG-3′,; (SEQ ID NO: 257)

200 μM dNTP, 2 mM MgCl₂, and 2.5 U taq DNA polymerase (Roche MolecularBiochemicals). The GeneAmp PCR System 9700 (Applied Biosystems) was usedto run the following program: 94° C. for 5 minutes; 40 cycles of (94° C.for 45 seconds, 55° C. for 45 seconds, 72° C. for 90 seconds); 72° C.for 10 minutes; cool to 4° C. The PCR products were purified withQIAquick 96 PCR Purification Kit (QIAGEN Inc.) according to themanufacturer's directions. All purified PCR products were sequenced withprimer 5′-TTACACTTTATGCTTCCG-3′ (SEQ ID NO: 258) using the ABI 3770Sequencer (Perkin Elmer) according to the manufacturer's directions.

5. Sequence Ranking

The peptide sequences that were translated from nucleotide sequences(from section 4C above) were correlated to ELISA data. The clones thatshowed high OD reading in the Ang-2 coated wells and low OD reading inthe BSA coated wells were considered more important. The sequences thatoccurred multiple times were also considered important. Twenty fourpeptide sequences from library 20, 26 peptide sequences from library 94,7 peptide sequences from library 25, 18 peptide sequences from library26, 6 peptide sequences from library 27, and 4 peptide sequences fromlibrary 99 were chosen for further analysis and peptibody generation.Additionally, eleven consensus sequences from libraries 20 and 94, threeconsensus sequences from libraries 26 and 99, and two from library 25were deduced and used to generate peptibodies. The peptibodies in Table7 were evaluated using the Neutralization ELISA protocol described inExample 10 herein. The results are shown in Table 7.

TABLE 7 Con4 Derived Affinity- Matured hAng-2:Tie2 Peptibody SequencePbs IC₅₀ (nM) (Seq Id No:) Con4-44 (C) 0.09 M-Fc-GGGGGAQ-PIRQEECDWDPWTCEHMWEV-LE (SEQ ID NO: 259) Con4-40 (C) 0.10 M-Fc-GGGGGAQ-TNIQEECEWDPWTCDHMPGK-LE (SEQ ID NO: 260) Con4-4 (C) 0.12 M-Fc-GGGGGAQ-WYEQDACEWDPWTCEHMAEV-LE (SEQ ID NO: 261) Con4-31 (C) 0.16 M-Fc-GGGGGAQ-NRLQEVCEWDPWTCEHMENV-LE (SEQ ID NO: 262) Con4-C5 (C) 0.16 M-Fc-GGGGGAQ-AATQEECEWDPWTCEHMPRS-LE (SEQ ID NO: 263) Con4-42 (C) 0.17 M-Fc-GGGGGAQ-LRHQEGCEWDPWTCEHMFDW-LE (SEQ ID NO: 264) Con4-35 (C) 0.18 M-Fc-GGGGGAQ-VPRQKDCEWDPWTCEHMYVG-LE (SEQ ID NO: 265) Con4-43 (C) 0.18 M-Fc-GGGGGAQ-SISHEECEWDPWTCEHMQVG-LE (SEQ ID NO: 266) Con4-49 (C) 0.19 M-Fc-GGGGGAQ-WAAQEECEWDPWTCEHMGRM-LE (SEQ ID NO: 267) Con4-27 (C) 0.22 M-Fc-GGGGGAQ-TWPQDKCEWDPWTCEHMGST-LE (SEQ ID NO: 268) Con4-48 (C) 0.26 M-Fc-GGGGGAQ-GHSQEECGWDPWTCEHMGTS-LE (SEQ ID NO: 269) Con4-46 (C) 0.26 M-Fc-GGGGGAQ-QHWQEECEWDPWTCDHMPSK-LE (SEQ ID NO: 270) Con4-41 (C) 0.26 M-Fc-GGGGGAQ-NVRQEKCEWDPWTCEHMPVR-LE (SEQ ID NO: 271) Con4-36 (C) 0.28 M-Fc-GGGGGAQ-KSGQVECNWDPWTCEHMPRN-LE (SEQ ID NO: 272) Con4-34 (C) 0.28 M-Fc-GGGGGAQ-VKTQEHCDWDPWTCEHMREW-LE (SEQ ID NO: 273) Con4-28 (C) 0.30 M-Fc-GGGGGAQ-AWGQEGCDWDPWTCEHMLPM-LE (SEQ ID NO: 274) Con4-39 (C) 0.30 M-Fc-GGGGGAQ-PVNQEDCEWDPWTCEHMPPM-LE (SEQ ID NO: 275) Con4-25 (C) 0.31 M-Fc-GGGGGAQ-RAPQEDCEWDPWTCAHMDIK-LE (SEQ ID NO: 276) Con4-50 (C) 0.38 M-Fc-GGGGGAQ-HGQNMECEWDPWTCEHMFRY-LE (SEQ ID NO: 277) Con4-38 (C) 0.40 M-Fc-GGGGGAQ-PRLQEECVWDPWTCEHMPLR-LE (SEQ ID NO: 278) Con4-29 (C) 0.41 M-Fc-GGGGGAQ-RTTQEKCEWDPWTCEHMESQ-LE (SEQ ID NO: 279) Con4-47 (C) 0.44 M-Fc-GGGGGAQ-QTSQEDCVWDPWTCDHMVSS-LE (SEQ ID NO: 280) Con4-20 (C) 0.48 M-Fc-GGGGGAQ-QVIGRPCEWDPWTCEHLEGL-LE (SEQ ID NO: 281) Con4-45 (C) 0.48 M-Fc-GGGGGAQ-WAQQEECAWDPWTCDHMVGL-LE (SEQ ID NO: 282) Con4-37 (C) 0.49 M-Fc-GGGGGAQ-LPGQEDCEWDPWTCEHMVRS-LE (SEQ ID NO: 283) Con4-33 (C) 0.52 M-Fc-GGGGGAQ-PMNQVECDWDPWTCEHMPRS-LE (SEQ ID NO: 284) AC2-Con4 (C) 0.52 M-Fc-GGGGGAQ-FGWSHGCEWDPWTCEHMGST-LE (SEQ ID NO: 285) Con4-32 (C) 0.75 M-Fc-GGGGGAQ-KSTQDDCDWDPWTCEHMVGP-LE (SEQ ID NO: 286) Con4-17 (C) 0.96 M-Fc-GGGGGAQ-GPRISTCQWDPWTCEHMDQL-LE (SEQ ID NO: 287) Con4-8 (C) 1.20 M-Fc-GGGGGAQ-STIGDMCEWDPWTCAHMQVD-LE (SEQ ID NO: 288) AC4-Con4 (C) 1.54 M-Fc-GGGGGAQ-VLGGQGCEWDPWTCRLLQGW-LE (SEQ ID NO: 289) Con4-1 (C) 2.47 M-Fc-GGGGGAQ-VLGGQGCQWDPWTCSHLEDG-LE (SEQ ID NO: 290) Con4-C1 (C) 2.75 M-Fc-GGGGGAQ-TTIGSMCEWDPWTCAHMQGG-LE (SEQ ID NO: 291) Con4-21 (C) 3.21 M-Fc-GGGGGAQ-TKGKSVCQWDPWTCSHMQSG-LE (SEQ ID NO: 292) Con4-C2 (C) 3.75 M-Fc-GGGGGAQ-TTIGSMCQWDPWTCAHMQGG-LE (SEQ ID NO: 293) Con4-18 (C) 4.80 M-Fc-GGGGGAQ-WVNEVVCEWDPWTCNHWDTP-LE (SEQ ID NO: 294) Con4-19 (C) 5.76 M-Fc-GGGGGAQ-VVQVGMCQWDPWTCKHMRLQ-LE (SEQ ID NO: 295) Con4-16 (C) 6.94 M-Fc-GGGGGAQ-AVGSQTCEWDPWTCAHLVEV-LE (SEQ ID NO: 296) Con4-11 (C) 9.70 M-Fc-GGGGGAQ-QGMKMFCEWDPWTCAHIVYR-LE (SEQ ID NO: 297) Con4-C4 (C) 9.80 M-Fc-GGGGGAQ-TTIGSMCQWDPWTCEHMQGG-LE (SEQ ID NO: 298) Con4-23 (C) 9.88 M-Fc-GGGGGAQ-TSQRVGCEWDPWTCQHLTYT-LE (SEQ ID NO: 299) Con4-15 (C) 15.00 M-Fc-GGGGGAQ-QWSWPPCEWDPWTCQTVWPS-LE (SEQ ID NO: 300) Con4-9 (C) 20.11 M-Fc-GGGGGAQ-GTSPSFCQWDPWTCSHMVQG-LE (SEQ ID NO: 301) Con4-10 (C) 86.61 M-Fc-GGGGGAQ-TQGLHQCEWDPWTCKVLWPS-LE (SEQ ID NO: 302) Con4-22 (C) 150.00M-Fc-GGGGGAQ- VWRSQVCQWDPWTCNLGGDW-LE (SEQ ID NO: 303) Con4-3 (C) 281.50M-Fc-GGGGGAQ- DKILEECQWDPWTCQFFYGA-LE (SEQ ID NO: 304) Con4-5 (C) NoInhibition M-Fc-GGGGGAQ- ATFARQCQWDPWTCALGGNW-LE (SEQ ID NO: 305)Con4-30 (C) No Inhibition M-Fc-GGGGGAQ- GPAQEECEWDPWTCEPLPLM-LE (SEQ IDNO: 306) Con4-26 (C) No Inhibition M-Fc-GGGGGAQ- RPEDMCSQWDPWTWHLQGYC-LE(SEQ ID NO: 307) Con4-7 (C) No Inhibition M-Fc-GGGGGAQ-LWQLAVCQWDPQTCDHMGAL-LE (SEQ ID NO: 308) Con4-12 (C) No InhibitionM-Fc-GGGGGAQ- TQLVSLCEWDPWTCRLLDGW-LE (SEQ ID NO: 309) Con4-13 (C) NoInhibition M-Fc-GGGGGAQ- MGGAGRCEWDPWTCQLLQGW-LE (SEQ ID NO: 310)Con4-14 (C) No Inhibition M-Fc-GGGGGAQ- MFLPNECQWDPWTCSNLPEA-LE (SEQ IDNO: 311) Con4-2 (C) No Inhibition M-Fc-GGGGGAQ- FGWSHGCEWDPWTCRLLQGW-LE(SEQ ID NO: 312) Con4-6 (C) No Inhibition M-Fc-GGGGGAQ-WPQTEGCQWDPWTCRLLHGW-LE (SEQ ID NO: 313) Con4-24 (C) No InhibitionM-Fc-GGGGGAQ- PDTRQGCQWDPWTCRLYGMW-LE (SEQ ID NO: 314) AC1-Con4 (C) NoInhibition M-Fc-GGGGGAQ- TWPQDKCEWDPWTCRLLQGW.LE (SEQ ID NO: 315)AC3-Con4 (C) No Inhibition M-Fc-GGGGGAQ- DKILEECEWDPWTCRILQGWLE (SEQ IDNO: 316) AC5-Con4 (C) No Inhibition M-Fc-GGGGGAQ-AATQEECEWDPWTCRLLQGW-LE (SEQ ID NO: 317) L1 Derived Affinity- MaturedhAng-2:Tie2 Peptibody Sequence Pbs IC₅₀ (nM) (Seq Id No:) L1-7(N) 0.03MGAQ- TNFMPMDDLEQRLYEQFILQQG- LEGGGGG-Fc (SEQ ID NO: 318) AC6-L1 (N)0.03 MGAQ- TNYKPLDELDATLYEHWILQHS LEGGGGG-Fc (SEQ ID NO: 319) L1-15 (N)0.04 MGAQ- QKYQPLDELDKTLYDQFMLQQG LEGGGGG-Fc (SEQ ID NO: 320) L1-2 (N)0.04 MGAQ- LNFTPLDELEQTLYEQWTLQQS LEGGGGG-Fc (SEQ ID NO: 321) L1-10 (N)0.05 MGAQ- QKFQPLDELEQTLYEQFMLQQA LEGGGGG-Fc (SEQ ID NO: 322) L1-13 (N)0.05 MGAQ- QEYEPLDELDETLYNQWMFHQR LEGGGGG-Fc (SEQ ID NO: 323) L1-5 (N)0.05 MGAQ- VKYKPLDELDEILYEQQTFQER LEGGGGG-Fc (SEQ ID NO: 324) L1-C2 (N)0.05 MGAQ- TKFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (SEQ ID NO: 325) L1-C3 (N)0.06 MGAQ- TNFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (SEQ ID NO: 326) L1-11 (N)0.07 MGAQ- QNFKPMDELEDTLYKQFLFQHS LEGGGGG-Fc (SEQ ID NO: 327) L1-17 (N)0.08 MGAQ- VKYKPLDELDEWLYHQFTLHHQ LEGGGGG-Fc (SEQ ID NO: 328) L1-12 (N)0.08 MGAQ- YKFTPLDDLEQTLYEQWTLQHV LEGGGGG-Fc (SEQ ID NO: 329) L1-1 (N)0.08 MGAQ- QNYKPLDELDATLYEHHFHYT LEGGGGG-Fc (SEQ ID NO: 330) L1-4 (N)0.08 MGAQ- VKFKPLDALEQTLYEHWMFQQA LEGGG43G-Fc (SEQ ID NO: 331) L1-20 (N)0.09 MGAQ- EDYMPLDALDAQLYEQFILLHG LEGGGGG-Fc (SEQ ID NO: 332) L1-22 (N)0.09 MGAQ- YKFNPMDELEQTLYEEFLFQHA LEGGGGG-Fc (SEQ ID NO: 333) L1-14 (N)0.11 MGAQ- SNFMPLDELEQTLYEQFMLQHQ LEGGGGG-Fc (SEQ ID NO: 334) L1-16 (N)0.11 MGAQ- QKFQPLDELEETLYKQWTLQQR LEGGGGG-Fc (SEQ ID NO: 335) L1-18 (N)0.16 MGAQ- QKFMPLDELDEILYEQFMFQQS LEGGGGG-Fc (SEQ ID NO: 336) L1-3 (N)0.16 MGAQ- TKFNPLDELEQTLYEQWTLQHQ LEGGGGG-Fc (SEQ ID NO: 337) L1-21 (N)0.17 MGAQ- HTFQPLDELEETLYYQWLYDQL LEGGGGG-Fc (SEQ ID NO: 338) L1-C1 (N)0.56 MGAQ- QKFKPLDELEQTLYEQWTLQQR LEGGGGG-Fc (SEQ ID NO: 339) L1-19 (N)1.26 MGAQ- QTFQPLDDLEEYLYEQWIRRYH LEGGGGG-Fc (SEQ ID NO: 340) L1-9 (N)1.62 MGAQ- SKFKPLDELEQTLYEQWTLQHA LEGGGGG-Fc (SEQ ID NO: 341) Con1Derived Affinity- Matured hAng-2:Tie2 Peptibody Sequence Pbs IC₅₀ (nM)(Seq Id No: Con1-4 (C) 1.68 M-Fc-GGGGGAQ- SGQLRPCEEIFGCGTQNLAL-LE (SEQID NO: 342) Con1-1 (C) 3.08 M-Fc-GGGGGAQ- AGGMRPYDGMLGWPNYDVQA-LE (SEQID NO: 343) Con1-6 (C) 8.60 M-Fc-GGGGGAQ- GQDLRPCEDMFGCGTKDWYG-LE (SEQID NO: 344) Con1-3 (C) 16.42 M-Fc-GGGGGAQ- APGQRPYDGMLGWPTYQRIV-LE (SEQID NO: 345) Con1-2 (C) No Inhibition M-Fc-GGGGGAQ-QTWDDPCMHILGPVTWRRCI-LE (SEQ ID NO: 346) Con1-5 (C) No InhibitionM-Fc-GGGGGAQ- FGDKRPLECMFGGPIQLCPR-LE (SEQ ID NO: 347) Parent: 26.00M-Fc-GGGGGAQ- Con1 (C) KRPCEEIFGGCTYQ-LE (SEQ ID NO: 348) 12-9 DerivedAffinity- Matured hAng-2:Tie2 Peptibody Sequence Pbs IC₅₀ (nM) (Seq IdNo:) 12-9-3 (C) 0.81 M-Fc-GGGGGAQ- LQEWCEGVEDPFTFGCEKQR-LE (SEQ ID NO:349) 12-9-7 (C) 0.93 M-Fc-GGGGGAQ- MLDYCEGMDDPFTFGCDKQM-LE (SEQ ID NO:350) 12-9-6 (C) 0.95 M-Fc-GGGGGAQ- HQEYCEGMEDPFTFGCEYQG-LE (SEQ ID NO:351) 12-9-C2 (C) 1.41 M-Fc-GGGGGAQ- LQDYCEGVEDPFTFGCENQR-LE (SEQ ID NO:352) 12-9-5 (C) 1.56 M-Fc-GGGGGAQ- LLDYCEGVQDPFTFGCENLD-LE (SEQ ID NO:353) 12-9-1 (C) 1.84 M-Fc-GGGGGAQ- GFEYCDGMEDPFTFGCDKQT-LE (SEQ ID NO:354) 12-94 (C) 2.05 M-Fc-GGGGGAQ- AQDYCEGMEDPFTFGCEMQK-LE (SEQ ID NO:355) 12-9-C1 (C) 2.68 M-Fc-GGGGGAQ- LQDYCEGVEDPFTFGCEKQR-LE (SEQ ID NO:356) 12-9-2 (C) 8.42 M-Fc-GGGGGAQ- KLEYCDGMEDPFTQGCDNQS-LE (SEQ ID NO:357) Parent: 15.00 M-Fc-GGGGGAQ- 12-9 (C) FDYCEGVEDPFTFGCDNH-LE (SEQ IDNO: 358)

EXAMPLE 9

Six samples of anti-Ang-2 peptibodies were tested for their bindingactivity to huAng-2 (R&D Systems, BNO12103A) on BIAcore. Protein G wasimmobilized to a CM5 chip according to the standard amine-couplingprotocol (BIAcore Inc.), and the peptibodies were then injected over aprotein G surface for capturing (RL˜100 Ru). To test binding betweenhAng-2 and the captured peptibody, 0.3 nM to 40 nM of huAng-2 wasinjected over the captured peptibody surfaces, and binding sensorgramswere analyzed using BIAevaluation 3.0 (BIAcore Inc.). Table 8 summarizesthe results of this experiment.

TABLE 8 Peptibody Lot # KD (M) ka (1/Ms) kd (1/s) Con4-44 (C) 0117022.1E−10 2.9E+05 5.9E−05 L1-7 (N) 022102 2.4E−10 3.7E+05 8.7E−05 L1-10(N) 021302 7.7E−10 1.5E+05 1.1E−04 L1-21 (N) 021802 2.4E−10 5.6E+051.4E−04 Con4 (C) 33456–77 3.8E−10 5.3E+05 2.0E−04 2xCon4 (C) 1K 0925013.4E−10 4.8E+05 1.6E−04

EXAMPLE 10

Neutralization ELISA

The human, murine, cyno, and rat Ang-2 and human and murine Ang-1conditioned media were diluted in DMEM/50 μg/ml BSA as follows:hAng-2—1:64 dilution; mAng-2—1:64 dilution; rat Ang-2—undiluted; cynoAng-2—1:32 dilution; hAng-1—1:4 dilution; and mAng-1—1:4 dilution.

The extent to which each of these conditioned media was diluted wasdetermined by their ability to bind 1 nM hTie²-Fc (provided as aTie-2-Fc molecule where the Tie-2 portion contains only the solubleextracellular portion of the molecule; R&D Systems, catalog number313-TI) at 50% of maximally achievable binding (i.e., plateau).Microtiter plates were coated with 100 μl of the diluted conditionedmedia. For Ang-2 neutralization ELISAs, candidate anti-Ang-2 peptibodieswere titrated from 62.5 nM to 0.015 pM in 4-fold dilutions in a solutionof PBS containing about 1% BSA and about 1 nM Tie-2 (provided as aTie-2-Fc molecule where the Tie-2 portion contains only the solubleextracellular portion of the molecule; R&D Systems, catalog number313-TI). For Ang-1 neutralization ELISAs, candidate anti-Ang-2peptibodies were titrated from 1000 nM to 0.2 pM in 4-fold dilutions ina solution of PBS containing about 1% BSA and about 1 nM Tie-2 (providedas a Tie-2-Fc molecule where the Tie-2 portion contains only the solubleextracellular portion of the molecule; R&D Systems, catalog number313-TI).

After about 100 microliters of the peptibody/Tie-2 solution was added toeach well, the plates were incubated overnight at room temperature, andthen washed five times in PBS containing about 0.1 percent Tween-20.After washing, about 100 microliters per well of anti-Tie-2 antibody(Pharningen Inc., catalog #557039) was added to a final concentration ofabout 1 microgram per ml, and the plates were incubated about 1 hour atroom temperature. Next, about 100 microliters per well of goatanti-mouse-IgG-HRP (Pierce Chemical Co., catalog #31432) was added at adilution of 1:10,000 in PBS containing about 1% BSA.

Plates were incubated at room temperature. for about 1 hour, after whichthey were washed five times with PBS containing about 0.1 percentTween-20. About 100 microliters per well of TMB substrate (SIGMA,catalog #T8665) was then added and blue color was allowed to develop.Absorbance was then read in a spectrophotomer at 370 nm. The results areset forth in Table 9 below.

TABLE 9 Peptibody-Mediated Neutralization of Angiopoietin:Tie2Interactions hAng-2 mAng-2 rAng-2 cAng-2 hAng-1 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀mAng-1 Peptibody (nM) (nM) (nM) (nM) (nM) IC₅₀ (nM) 2xCon4 (C) 0.0260.035 0.024 0.047 3.0 3.2 Con4 (C) 0.197 0.289 0.236 0.540 200 300Con4-44 (C) 0.08 0.16 0.22 — 43 — Con4-40 (C) 0.20 0.27 0.35 — >1000 —L1-7 (N) 0.046 0.063 0.035 0.108 >1000 >1000 L1-21 (N) 0.179 0.249 0.2040.608 >1000 >1000 L1-10 (N) 0.06 0.06 0.06 — >1000 —

EXAMPLE 11 PK Study

Study Design

Male CD-1 mice, weighing 20-30 g, were randomly divided into eachpeptibody treatment group (2xCon-4-C, L1-7-N, and L1-21-N). Animalsreceived a single IV bolus (n=38/group) or a single SC administration of50 μg peptibody (n=34/group). The injections were done via the tail veinand under the skin over the shoulders for IV and SC administrations,respectively.

Blood Sampling and Analytical Methods

Blood samples were collected for each anti-Ang-2 peptibody concentrationmeasurement predose, and at 1, 2, 4, 8, 16, 24, 48, 72, 96, 120, 144,168, 216, 264, 312, and 336 hours after dose administration for the SCand IV groups. Additional samples were collected at 5 and 30 minutespostdose for IV groups. Two animals were bled per time point, andanimals were sacrificed after sampling. Blood (approximately 0.50 mL)was collected from a cardiac puncture into polypropylene microtainer®)serum separator tubes. Samples were kept on ice for approximately 20minutes or until clot formation occurred. Serum was separated from theblood samples by centrifugation for approximately 10 minutes at 2-8° C.,and stored at approximately −70° C. until assayed. Samples were measuredusing a verified time resolved fluorescence (TRF) assay with a lowerlimit of quantification (LLOQ) of 100 ng/mL. NUNC fluoroMaxisorpmicrotiter plates were coated with recombinant mouse Ang-2 protein. Theplates were then blocked with a protein solution to reduce nonspecificbinding. Standards, quality controls and unknown samples were preparedin 10% mouse serum assay buffer and pipetted into wells of microtiterplates. The peptibodies were bound specifically to the immobilizedAng-2. After washing away any unbound substances (Kirkegaard & PerryLaboratories Inc.), a biotinylated goat anti-Human IgG (H+L) monoclonalantibody (Jackson ImmunoResearch Laboratories Inc.) was added to thewells. Following a wash step to remove any unbound biotinylatedmonoclonal antibody, europium labelled streptavidin was added to thewells. After washing off the unbound streptavidin europium, the boundeuropium was released from the streptavidin with an acidic solutionpipetted into each well. Fluorescent signal was generated and read inthe Wallac's fluorometric reader. The assay range for the analysis ofanti-Ang-2 peptibody in mouse serum is 0.078-5 μg/mL.

Pharmacokinetic Analysis

The composite mean concentration-time data for each group were subjectedto noncompartmental analysis using WinNonlin Professional (Version 3.3,Pharsight Corp., Mountain View, Calif.). The nominal sampling times wereused for PK analysis, as samples were collected within 10% of thenominal time. All concentration values less than the LLOQ were set tozero before PK analysis. The following PK parameters were estimated:

-   -   Terminal half-life (t_(1/2)) was calculated as

${t_{1/2} = \frac{\ln(2)}{k_{el}}},$

-   -    where k_(el) was the first-order terminal rate constant        estimated via linear regression of the terminal log-linear decay        phase.    -   The area under the serum concentration-time curve        (AUC_((0-last))) was estimated using the linear/log trapezoidal        method from time 0 to last, the time of the last quantifiable        concentration (C_(last)).    -   The area under the curve from time 0 to infinity (AUC_((0-∞)))        was estimated as the sum of the corresponding AUC_((0-last)) and        the predicted C_(last/k) _(el) values:

${AUC}_{({0 - \infty})} = {{AUC}_{({0 - {last}})} + \frac{{Predicted}\mspace{14mu} C_{last}}{k_{el}}}$

-   -   Absolute bioavailability (F) after SC administration was        calculated as:

$F = {\frac{{AUC}_{{({0 \cdot \infty})}{SC}}}{{AUC}_{{({0 - \infty})}{IV}}} \times 100}$

The results are set forth in FIG. 2.

EXAMPLE 12

Female nude mice were injected subcutaneously with 1×10⁷ A431 cells onstudy day 0. At day 3, the Ang-2 peptibody 2xCon4-C was administeredsubcutaneously at a dose of 200 μg/mouse/day. Tumor volumes and bodyweights were recorded at regular intervals, as shown in the figure.Significant differences in tumor growth were observed between the Ang-2peptibody-treated group versus vehicle control and control peptibody(p<0.0001 vs. each control using repeated measure ANOVA, with Scheffe'spost hoc test). Treatment with this peptibody had no significant effecton body weights. The results are set forth in FIG. 3.

EXAMPLE 13 A431 In Vitro Growth Curve

A431 cells were seeded in 96-well tissue culture plates at 2000 cellsper well, in 200 μl of DMEM supplemented with 10% fetal bovine serum(FBS). The medium was then aspirated 16 hours post seeding. Thefollowing were then added back into the wells and set up in triplicate:100 μl per well of DMEM, 10% FBS, 1 mg/ml negative control peptibody4883 or peptibody TN8-Con4. The same set-ups were repeated on 5 plates.Medium from one plate was aspirated at 24, 48, 72, 96, and 120 hourspost treatment. One hundred μl of 10% trichloroacetic acid (TCA) perwell were then added, and the plates were then stored at 4° C. All ofthe plates were collected when the last plate had been in 10% TCA for aminimum of 4 hours. The 10% TCA was shaken out, and the wells wererinsed 5 times with tap water. The cells were then stained with 100 μl0.4% sulforhodamine B (Sigma S-9012) in 1% acetic acid (Sigma A-6283)for 10 minutes at room temperature, and then washed 5 times with 1%acetic acid. The plates were then air dried. The dye was solubilizedwith 300 μl 20 mM unbuffered Tris (pH>10) for 2 hours on a rotaryshaker. Optical density (OD) was then read at 540 nm on a microtiterplate reader. The results are set forth in FIG. 4.

EXAMPLE 14

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. At day 3, the Ang-2 peptibodiesL1-7-N, L1-21-N, Con4-C, and 2xCon4-C were administered subcutaneouslyat the dose of 14 μg/mouse, twice a week. Anti-Ang-2 antibody Ab536, 47μg/mouse, three times a week, was administered as a positive control.Tumor volumes and body weights were recorded at regular intervals.

Significant differences in tumor growth were observed between each oneof the Ang-2 peptibody treated group versus vehicle control and controlpeptibody (p<0.0001 vs. each control using repeated measure ANOVA, withScheffe's post hoc test). Treatment with these peptibodies had nosignificant effect on body weights (results not shown). The results areset forth in FIG. 5.

EXAMPLE 15

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. At day 3, the Ang-2 peptibody2xCon4-C was administered subcutaneously at the doses of 14, 2.8, and0.56 μg/mouse, twice a week. Tumor volumes and body weights wererecorded at regular intervals, as shown. Significant differences intumor growth were observed between the two higher doses of the Ang-2peptibody treated group versus vehicle control and control peptibody(p=0.003 for the intermediate dose and p<0.0001 for the high dose, usingrepeated measure ANOVA, with Scheffe's post hoc test). Treatment withthese peptibodies had no significant effect on body weights. The dashedline represent a reduction of the total n of the group, from 10 to 9mice, due to the death of one mouse for unknown reasons. The results areset forth in FIG. 6.

EXAMPLE 16 Anti-Ang-2 peptibodies vs. Colo-205 Xenograft Tumors

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. At day 3, Ang-2 peptibody 2xCon4-Cor control peptibody were administered subcutaneously at the dose of 350μg/day. Tumors from groups treated with control peptibody (as describedin Table 5) were harvested either at Day 14 (size-matched control) orDay 18 (time-matched control). Tumors from 2xCon4(C) treated group werethen harvested at Day 18. Tumor volumes were recorded at regularintervals, as shown. Significant differences in tumor growth wereobserved between the time-matched control group and the 2xCon4-C treatedgroup (p=0.0154 by repeated measure ANOVA, with Scheffe's post hoctest). Treatment with these peptibodies had no significant effect onbody weight.

Tumors prepared for image analysis were bisected coronally and one-halfsnap frozen in OCT (Sakura Finetek USA Inc., Torrance, Calif.).Cryo-sections were immunohistochemically stained using anti-mouse CD31(catalogue #553370, BD PharMingen, San Diego, Calif.) at 2 μg/ml, withDAB as the chromogen. The tumor sections were digitally photographed at20× objective magnification. Four “compass-point” fields per tumor werecaptured, with ten tumors per treatment group. A MetaMorph (UniversalImaging Corporation, Downington, Pa.) image analysis system was used tothreshold for the CD31 stained blood vessels within the images. Theareas of CD31 positive staining were expressed as a ratio of the totaltumor tissue within each field. The results are set forth in FIG. 7.

EXAMPLE 17

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. Treatment with 350 μg/mouse, s.c.twice a week, of the Ang-2 peptibody 2xCon4-C, or equivalent controlpeptibody started either at study day 3, 10 or 15. Tumor volumes andbody weights were recorded at regular intervals. Significant differencesin tumor growth were observed between all Ang-2 peptibody treated groupversus vehicle control (p=0.089 for day 15 group and p<0.0001 for day 3and 10 groups, using repeated measure ANOVA, with Scheffe's post hoctest). Treatment with these peptibodies had no significant effect onbody weights. The results are set forth in FIG. 8 (body weights notshown).

EXAMPLE 18

A summary of complete response (CR) rates was obtained using antibodyAb536 at 47 μg/female nude mouse, administered intraperitoneally threetimes a week, or with peptibody 2xCon4(C), given subcutaneously atmultiple dosing schedules in different long term studies (≧10 weeks ofdosing) in both the A431 and Colo-205 xenograft models. CR as usedherein refers to an outcome in which no measurable tumor remainedfollowing treatment. The results are set forth in FIG. 9.

EXAMPLE 19

a) Combination of Pb with Taxotere in the Colo-205 Tumor Model

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. At study day 14, treatments werestarted with a) 350 μg/mouse, s.c. twice a week, of the Ang-2 peptibody2xCon4-C, b) 20 mg/kg qwx3 i.p. of taxotere, or c) a combination ofboth. Tumor volumes and body weights were recorded at regular intervals.Significant differences in tumor growth were observed between alltreatment groups versus vehicle control (p<0.0001 using repeated measureANOVA, with Scheffe's post hoc test). In addition, the combinationtherapy group was significantly different than either one of themonotherapy agents (p<0.0001 vs. 2xCon-4-C and p=0.0122 vs taxotere).The dashed line represents a reduction of the total n of the group, from10 to 9 mice, due to the death of one mouse for unknown reasons.Treatment with these peptibodies had no significant effect on bodyweights. The results are set forth in FIG. 10 a.

b) Combination of Pb with 5-FU in the Colo-205 Tumor Model

Female nude mice were injected subcutaneously with 2×10⁶ Colo-205 cellsplus Matrigel (2:1) on study day 0. At study day 14 started treatmentswith a) 350 μg/mouse, s.c. twice a week, of the Ang-2 peptibody2xCon4-C, b) 50 mg/kg qdx5 i.p. of 5-FU, or c) a combination of both.Tumor volumes and body weights were recorded at regular intervals, asshown.

Significant differences in tumor growth were observed between alltreatment groups versus vehicle control (p<0.0001 using repeated measureANOVA, with Scheffe's post hoc test). In addition, the combinationtherapy group was significantly different than either one of themonotherapy agents (p=0.0375 vs. 2xCon-4-C and p=0.0453 vs. 5-FU). Atransient reduction in body weight was observed in the 5-FU treatedgroup (18% at study day 20) as well as with the combination therapygroup (16% at study day 20), followed by a complete recovery of the bodyweights. The results are set forth in FIG. 10 b.

EXAMPLE 20

Adjuvant Arthritis Model

Male Lewis rats (120-130 g, Charles River, Wilmington Mass.) were housedtwo per filter-capped cage in an environmentally controlled room(temperature 23±2° C., relative humidity 50±20%) on a 12-hourlight/darkcycle. Animals were fed a commercial rodent chow (Formulation 8640; TekLab, Madison, Wis.) and received filter-purified tap water ad libitum.Dietary calcium and phosphorus contents were 1.2% and 1.0%,respectively.

Adjuvant arthritis was induced by a single 0.5 mg injection ofheat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories,Detroit, Mich.) suspended in 0.05 mL paraffin oil (Crescent ChemicalCo., Hauppauge, N.Y.) intradermally at the base of the tail. Theclinical onset of arthritis was at day 9 as indicated by hind pawswelling and ambulatory difficulties. Except in the 2xCon4(c) treatedgroup (which was treated from Day 1 after immunization), treatments weregiven as daily subcutaneous injections beginning at day 9 afterimmunization (prior to onset of arthritis) and continuing through day18.

Clinical Monitoring of Adjuvant Arthritis.

The progression of inflammation was assessed clinically by theintermittent measurement of hind paw volume using water plethysmographyaccording to the methods described by Feige et al., Cellular Molec. LifeSci., 57:1457-1470 (2000). Inhibition of paw inflammation was calculatedbased on the area under the curve (AUC) using the trapezoidal ruleaccording to the formula:[1−{(Treated AdA)−normal)/(Untreated AdA−normal)}]×100

In addition, total body weight was determined daily during the 9-daytreatment regimen as a supplemental endpoint because body weight losshas been shown to parallel the progression of joint inflammation in thisarthritis model. Animals were sacrificed under CO₂ on day 18.

Loss of bone mineral density (BMD) was examined at necropsy (day 18 postimmunization). Hind paws were removed at the fur line (just proximal tothe ankle (hock)), immersed in 70% ethanol, and then scanned inhorizontal orientation using a fan beam X-ray densitometer (ModelQDR4500A; Hologic, Waltham, Mass.). See Feige et al., supra. After thescan, a rectangular box (29×25 mm) centered at the calcaneus waspositioned to delineate the site to be analyzed, and proprietaryalgorithms (Hologic software) calculated bone area, bone mineralcontent, and bone mineral density.

All results were expressed as the mean ± standard error. A p value of0.05 was used to delineate significant differences between groups. AKruskal-Wallis ANOVA and a Mann-Whitney U. test using commercialstatistical software (Statsoft v3.0; Statsoft, Tulsa, Okla.) wereperformed on the clinical data (continuous variables).

The results are set forth in FIG. 11 a, 11 b, and 11 c, respectively.

EXAMPLE 21 Corneal Angiogenesis Model Effect of CON4(C) on VEGF-InducedAngiogenesis in Rats

Ang-2 peptibody CON4(C) was evaluated in the corneal model ofangiogenesis in rats. Angiogenesis was induced by implanting a VEGF- (orBSA control) soaked nylon disc into the corneal stroma (n=8/group).Peptibody TN8CON4-C was administered by sub-cutaneous injection at 1.0or 0.1 mg/rat/day for seven days. Two other groups of animals weretreated with the same dose of negative control peptibody 4883. Allgroups were pre-treated with a single loading dose of either 3.0 or 0.3mg that was three times the maintenance dose of 1.0 or 0.1 mg (seefigure). After seven days of treatment, two vascular endpoints weredetermined from each digital image of the rat cornea: the number ofvessels intersecting the mid-point between the disc and the limbus, andthe blood vessel area. Treatment with TN8CON4-C significantly inhibitedVEGF-induced angiogenesis in a dose-dependent manner (p<0.04), whereastreatment with the control peptibody had no significant effect on eitherend-point. There was no evidence of overt toxicity based on body weightsof the treated animals. The results are set forth in FIG. 12.

EXAMPLE 22 Epitope Mapping

Full-length (amino acids 1-495), N-terminal (amino acids 1-254) andC-terminal (amino acids 255-495) human Ang-2 (hAng-2) proteins werecloned into a CMV-driven mammalian expression vector with C-terminal6xHis tags. The three resultant constructs plus a vector control weretransiently expressed into 293T cells. Conditioned media were thencollected from the transfected cells, and the expression level of Ang-2in the media was estimated by anti-6xhis ELISA and Western blotting.

The binding epitope of anti-Ang-2 antibodies and peptibodies wasdetermined by their ability to bind the three versions of human hAng-2by ELISA according to the following protocol: a high-binding 96-wellassay plate was coated with 100 μl of conditioned media per well, andincubated at 37° C. for 1 hour. Conditioned media was aspirated, and theplate was blocked with 200 μl per well of 5% BSA in PBS at roomtemperature for 1 hour. The blocking solution was then aspirated. 100 μlper well of antibody, peptibody, or Tie²-Fc was added at 1 μg/ml in 1%BSA in PBS, and incubated at room temperature for 1 hour. The wells werewashed 4 times with 200 μl of 0.1% Tween in PBS. 100 μl per well ofHRP-conjugated goat anti-human IgG or goat anti-mouse IgG were added,and incubated at room temperature for 45 minutes. The wells were thenwashed with 200 μl of 0.1% Tween in PBS 4 times. 100 μl per well of TMBsubstrate was then added. O.D. was read at 370 nm.

The results are set forth in FIG. 13 a, FIG. 13 b, and FIG. 13 c.

EXAMPLE 23

Due to certain sensitivity limitations inherent in the BiaCore assay,binding affinity was also evaluated using a Sepidyne KinExA assay.

Binding of 2xCON4-C (Pb5714) to huAng-2 was tested on KinExA (Sapidyne,Boise, Id.). Reacti-Gel 6× beads (Pierce, Rockford, Ill.) werepre-coated with huAng-2 and blocked with BSA. 10 pM and 30 pM of2xCON4-C samples were incubated with various concentrations (0.3 pM-3nM) of huAng-2 at room temperature for 8 hours before run through thehuAng-2-coated beads. The amount of the bead-bound peptibody wasquantified by fluorescent (Cy5) labeled goat anti-human-Fc antibody(Jackson Immuno Research, West Grove, Pa.). The binding signal isproportional to the concentration of free peptibody at equilibrium.

The dissociation equilibrium constant (K_(D)) was obtained fromnonlinear regression of the competition curves using a dual-curveone-site homogeneous binding model (KinEx™ software). K_(D) was thendetermined to be approximately 2 pM for 2xCON4-C binding with huAng-2.

As is shown in FIG. 14, using the KinExA assay peptibody 2xCon4 wasshown to have ˜2 pM affinity for hAng-2.

EXAMPLE 24 Pegylated Peptides

L1-7 peptide was synthesized with a 431 ABI synthesizer using a standardcoupling protocol and double coupling from residue 14 (met) to theN-term residue 1 (Cys), numbering from the N-terminus to the C-terminus.

Conjugation of L1-7 Peptide with Methoxy-poly(ethyleneglycol)-maleimide; MW: 5 KDa; termed “mPEG5K-(L1-7 Peptide)”

A solution of 0.8 mg of L1-7 peptide in 400 μL of buffer 1 (20 mMphosphate, 5 mM EDTA, pH 6.5) was treated with 13.5 mg ofmethoxy-poly(ethylene glycol)-maleimide (MW=5 KDa; Shearwater Corp.);0.27 ml of a 50.0 mg/mL solution in buffer 1. The reaction mixture wasincubated at 4° C. overnight, then diluted with 1.6 mL of buffer A (20mM Tris hydrochloride, pH 7.2) and dialyzed in a Slide-A-Lyzer cassette(3500 MWCO, Pierce) against the same buffer. The dialyzed reactionmixture was purified by ion exchange chromatography on a 1.0 mL HiTrap QSepharose HP column (Amersham Biosciences Corp.). The product peak waseluted in two 1.0 mL fractions via a gradient from 100% buffer A to 100%buffer B (buffer A+0.5 M NaCl) over 40 column volumes. The combinedproduct fractions were concentrated to 250 μL containing 0.23 mgprotein/mL with a Microsep 1K Centrifugal Device (Pall Life Sciences).

Conjugation of L1-7 Peptide with 1.11-bis-maleimidotetraethyleneglycol;termed “PEO4(L1-7 Peptide)₂”

A solution of 1.0 mg of L1-7 peptide in 500 μL of buffer 1 (20 mMphosphate, 5 mM EDTA, pH 6.5) was treated with 0.0375 mg of 1,11-bis-maleimidotetraethyleneglycol (Pierce) (0.375 mL of a 0.1 mg/mLsolution in buffer 1). The reaction mixture was incubated at 4° C. for3.33 hrs, then dialyzed in a Slide-A-Lyzer cassette (3500 MWCO, Pierce)against buffer A (20 mM Tris hydrochloride, pH 7.2). The dialyzedreaction mixture was purified by ion exchange chromatography on a 1.0 mLHiTrap Q Sepharose HP column (Amersham Biosciences Corp.). The dimericproduct peak was eluted in three 1.0 mL fractions via a gradient from100% buffer A to 100% buffer B (buffer A+0.5 M NaCl) over 40 columnvolumes. The combined product fractions were concentrated to 550 μLcontaining 0.12 mg protein/mL with a Microsep 1K Centrifugal Device(Pall Life Sciences).

Conjugation of L1-7 Peptide with Poly(ethylene glycol)-bis-maleimide: MW3.4 KDa; termed “PEG3.4K(L1-7 Peptide)₂”

A solution of 3.0 mg of L1-7 Peptide in 1.5 mL of buffer 1 (20 mMphosphate, 5 nM EDTA, pH 6.5) was treated with 1.125 mg of poly(ethyleneglycol)-bis-maleimide (MW=3.4 KDa, Shearwater Corp.); 0.563 mL of a 2.0mg/mL solution in buffer 1. The reaction mixture was incubated at 4° C.for overnight, then dialyzed in a Slide-A-Lyzer cassette (3500 MWCO,Pierce) against buffer A (20 mM Tris hydrochloride, pH 7.2). Thedialyzed reaction mixture was purified by ion exchange chromatography ona 5.0 mL HiTrap Q Sepharose HP column (Amersham Biosciences Corp.). Theproduct peak was eluted in three 3.0 mL fractions via a gradient from100% buffer A to 100% buffer B (buffer A+0.5 M NaCl) over 40 columnvolumes. The combined product fractions were concentrated to 850 μLcontaining 0.24 mg protein/mL with two Microsep 1K Centrifugal Devices(Pall Life Sciences).

MALDI-TOF mass spectroscopy results were as follows:

Sample# Identity Exp. MS Obs. MS 1 L1-7 (unPEGylated Peptide) 3,5453,538.7 2 mPEG5K-(L1-7 Peptide) 8,500 8,851 3 PEO4(L1-7 Peptide)₂ 7,4437,446.29 4 PEG3.4K(L1-7 Peptide)₂ 10,550 10,552 6,882.61 3,550.13

It will be appreciated that the subscripted “₂” for the PEG3.4K(L1-7Peptide) and PEO4(L1-7 Peptide) indicates that there are two peptidesper polymer chain, one located on each end of the polymer.

IC₅₀ Determination

The IC₅₀ for inhibition of hAng-2:hTie²-Fc interaction for the L1-7 freeand PEGylated peptides were determined by the Neutralization ELISA asdescribed in Example 2. For the Neutralization ELISA, microtiter platesto which human Ang-2 polypeptide was bound were prepared as described inExample 2 for the Affinity ELISA. Candidate anti-Ang-2 L1-7 PEGylatedand Free petides were titrated from 1000 nM to 0.2 pM in 4-folddilutions in a solution of PBS containing about 1% BSA and about 1 nMTie-2 (provided as a Tie-2-Fc molecule where the Tie-2 portion containsonly the soluble extracellular portion of the molecule; R&D Systems,catalog number 313-TI). After about 100 microliters of theantibody/Tie-2 solution was added to each well, the plates wereincubated overnight at room temperature, and then washed five times inPBS containing about 0.1 percent Tween-20. After washing, about 100microliters per well of anti-Tie-2 antibody (Pharmingen Inc., catalog#557039) was added to a final concentration of about 1 microgram per ml,and the plates were incubated about 1 hour at room temperature. Next,about 100 microliters per well of goat anti-mouse-IgG-HRP (PierceChemical CO., catalog #31432) was added at a dilution of 1:10,000 in PBScontaining about 1 percent BSA. Plates were incubated at roomtemperature for about 1 hour, after which they were washed five timeswith PBS containing about 0.1 percent Tween-20. About 100 microlitersper well of TMB substrate (described above) was then added and color wasallowed to develop. Absorbance was then read in a spectrophotomer at 370nm.

-   -   L1-7 peptides (C-GGGGG-AQ-TNFMPMDDLEQRLYEQFILQQG-LE) (SEQ ID        NO: 359) included: an N-terminal Cysteine for coupling to PEG;        and a 5Gly linker. AQ and LE flanking sequences were present        both in the original phage clone and in the peptibody. The        hAng-2:Tie² Inhibition IC₅₀ results were as follows:

Peptide IC₅₀ (nM) L1-7 Peptide 0.49 mPEG5K-(L1-7 Peptide) 11.7 PEO4(L1-7Peptide)₂ 0.064 PEG3.4K(L1-7 Peptide)₂ 0.058

EXAMPLE 25

The effect of Ang-2 peptibody 2xCon4(C) on rat adjuvant arthritis wasstudied, administered beginning either at the time of immunization (day0) or immediately prior to onset of clinical symptoms of arthritis (day8). Ang-2 peptibody 2xCon4(C) was also studied in combination with aPEGylated TNF inhibitor, pegsunercept (PEG sTNF-R1).

Male Lewis rats (200-250 g) were obtained from Charles River(Wilmington, Mass.). Arthritis was induced using a single intradermalinjection at the base of the tail of 0.5 mg of heat-killed Mycobacteriumtuberculosis H37Ra, termed “MTb” (Difco Laboratories, Detroit, Mich.)suspended in 0.05 ml paraffin oil (Crescent Chemical Co., Hauppauge,N.Y.) on day 0. Treatments of PEG sTNF-R1 (30 mg/ml, given at 1 mg/kg),peptibody 2xCon4(C) (30.8 mg/ml, given at 1 mg/rat) or vehicle (PBS)were administered subcutaneously as outlined in the table below.

Group # MTb Group PEGsTnF-R1 or PBS 2xCon4(C) or PBS N 1 − NormalControl — — 6 2 + Arthritis Control — — 6 3 + PBS Control PBS day 0-17PBS day 0-17 6 4 + PEG sTNF-R1 PEG sTNF-R1 day 0-17 PBS day 0-17 6 5 +2xCon4(C) PBS day 0-17 2xCon4(C) day 0-17 6 6 + PEG sTNF-R1 + 2xCon4(C)PEG sTNF-R1 day 0-17 2xCon4(C) day 0-17 6 7 + PBS Control PBS day 8-17PBS day 8-17 6 8 + PEG sTNF-R1 PEG sTNF-R1 day 8-17 PBS day 8-17 6 9 +2xCon4(C) PBS day 8-17 2xCon4(C) day 8-17 6 10 + PEG sTNF-R1 + 2xCon4(C)PEG sTNF-R1 day 8-17 2xCon4(C) day 8-17 6 11 + PEG sTNF-R1 + 2xCon4(C)PEG sTNF-R1 day 8-17 2xCon4(C) day 0-17 6

A less than maximally effective dose of 1 mg/kg of PEG sTNF-R1 waschosen in order to allow assessment of potential additive effects oftherapy with peptibody 2xCon4(C). Hind paw volume was assessedclinically by daily measurement of hind paw volume using waterplethysmography (Feige et. al, Cell Mol Life Sci, 57:1457-1470 (2000)).Inhibition of paw inflammation was calculated based on area under thecurve (AUC) according to the formula:[(AUC Arthritic AdA−AUC Treated AdA)/AUC Arthritic AdA]×100

Total body weight was determined daily from day 9 to day 18 as asupplemental endpoint because body weight loss has been shown toparallel the progression of joint inflammation in this arthritis model.

In FIG. 16, results were expressed as the mean ± standard error. A pvalue of 0.05 was used to delineate significant differences betweengroups. A Kruskal-Wallis ANOVA and a Mann-Whitney U test with noadjustment for multiplicity of testing was performed on the clinicaldata (continuous variables).

Body Weight: Groups given either peptibody 2xCon4(C) or PEG sTNF-R1alone for any time period either lost or did not gain weight during theexperiment. Animals given combination therapy with peptibody 2xCon4(C)and PEG sTNF-R1 from days 8-17 maintained but did not gain weight.However, arthritic animals which received peptibody 2xCon4(C) on days0-17 together with PEG sTNF-R1 given in either time course (days 0-17 ordays 8-17) gained weight over the course of the experiment.

The results are set forth in FIGS. 15, 16, and 17.

1. A method for treating an inflammatory disease which comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a peptide or peptibody capable of binding Ang-2, wherein thepeptide or peptibody comprises at least one amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, and SEQ ID NO: 119 to SEQ IDNO: 142, inclusive, and physiologically acceptable salts thereof.
 2. Amethod for treating an inflammatory disease which comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a composition of matter of the formula:(X¹)_(a)-F¹-(X₂)_(b) and multimers thereof, wherein: F¹ is a vehicle; X¹X² are each independently selected from -(L¹)_(c)-P¹;-(L¹)_(c)-P¹-(L²)_(d)-P²; -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³; and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴; wherein a, b, c, d, eand fare each independently 0 or 1, provided that at least one of a andb is 1; wherein L¹, L², L³, and L⁴ are each independently linkers; andwherein one or more of P¹, P², P³, and P⁴ each independently comprise apeptide having an amino acid sequence selected from the group consistingof SEQ ID NO: 6 and SEQ ID NOS: 119-142, inclusive; and physiologicallyacceptable salts thereof.
 3. The method according to claim 1 furthercomprising administering at least one anti-inflammatory agent.
 4. Themethod according to claim 3 wherein the anti-inflammatory agentcomprises at least one of a disease modifying anti-rheumatic drug(DMARD), slow acting anti-rheumatic drug (SAARD), and non-steroidalanti-rheumatic drug (NSAID).
 5. The method according to claim 3 whereinthe anti-inflammatory agent comprises at least one of methotrexate, aTNF inhibitor, a IL-1 inhibitor, a TACE inhibitor, a COX-2 inhibitor,and a P-38 inhibitor.
 6. The method according to claim 5 wherein the TNFinhibitor comprises at least one of etanercept, adalimumab, pegsunerceptsTNF-R1, onercept, and infliximab.
 7. The method according to claim 5wherein said IL-1 inhibitor is at least one of anakinra, IL-1 TRAP, IL-1antibody, and soluble IL-1 receptor.
 8. The method according to claim 3wherein the administration is concurrent administration.
 9. The methodaccording to claim 3 wherein the administration is non-concurrentadministration.